Compositions for analyzing proteins

ABSTRACT

Methods, compositions and kits are disclosed for determining one or more target polypeptides in a sample where the target polypeptides have undergone a post-translational modification.

CROSS REFERENCE TO RELATED PATENT APPLICATION

This application is a continuation of co-pending application U.S. patentapplication Ser. No. 11/837,427, filed Aug. 10, 2007, which is adivisional application of U.S. patent application Ser. No. 10/154,042,filed May 21, 2002, now U.S. Pat. No. 7,255,999, which claims priorityto U.S. Provisional Application Ser. No. 60/292,548, filed May 21, 2001,and U.S. Provisional Application Ser. No. 60/334,901, filed Oct. 24,2001, all of which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

The present invention relates to separable compositions, methods, andkits for use in detection and quantitation of polypeptides. Theinvention finds particular application to the area of multiplexed assaysfor polypeptides including proteins involved in post-translationalactivity.

The need to determine many analytes including polypeptides and nucleicacid sequences in blood or other biological fluids has becomeincreasingly apparent in many branches of medicine. Most multi-analyteassays, such as assays in the genomics area that detect multiple nucleicacid sequences, involve multiple steps, have poor sensitivity, a limiteddynamic range (typically on the order of 2 to 100-fold differences, andsome require sophisticated instrumentation. Some of the known classicalmethods for multianalyte assays include the following:

a. The use of two different radioisotope labels to distinguish twodifferent analytes.

b. The use of two or more different fluorescent labels to distinguishtwo or more analytes.

c. The use of lanthanide chelates where both lifetime and wavelength areused to distinguish two or more analytes.

d. The use of fluorescent and chemiluminescent labels to distinguish twoor more analytes.

e. The use of two different enzymes to distinguish two or more analytes.

f. The use of enzyme and acridinium esters to distinguish two or moreanalytes.

g. Spatial resolution of different analytes, for example on arrays, toidentify and quantify multiple analytes.

h. The use of acridinium ester labels where lifetime or dioxetanoneformation is used to quantify two different viral targets.

Proteomics has come of interest over the last few years. Whileproteomics is more complex than genomics, the study of proteins givesmore accurate pictures of cell biology than studying mRNA. The field ofproteomics is very broad and involves areas such as, for example,protein profiling by the use of two-dimensional gel electrophoresis andmass spectrometry to study proteins expressed in the cell,protein-protein interaction using yeast two-hybrid method, pathwayanalysis to understand signal transduction and other complex cellprocesses, large scale protein folding and 3-D structure studies andhigh-throughput expression and purification of proteins, cellularexpression during metabolism, mitosis, meiosis, in response to anexternal stimulus, e.g., drug, virus, change in physical or chemicalcondition, involving excess or deficient nutrients and cofactors,stress, aging, presence of particular strains of an organism andidentifying the organism and strain, multiple drug resistance,protein-DNA interactions, protein peptide interactions, and the like. Itis necessary to have a means for identifying a large number of protein'sin a single sample, as well as providing some quantitation of thedifferent proteins being detected.

As the human genome is elucidated, there will be numerous opportunitiesfor performing diagnostic procedures relating to the coding sequences ofgenes. One major function of genes is to generate proteins, which play amajor role in the work carried out in a cell. Because the proteinfunctions in a cell are dynamic, the structure, concentration, location,and so forth of a particular protein at a particular point in time isconstantly changing. Analysis of protein expression patterns is thesubject of ongoing genomics projects. Studies of physiologically activeforms of proteins and their spatial and temporal interaction in the cellIs an important aspect of the overall study.

One post-translational modification of proteins is the addition orremoval of phosphate groups. Protein phosphorylation andde-phosphorylation reactions have been established as major componentsof metabolic regulation and signal transduction pathways. Variations inprotein phosphorylation provide the predominant means of enzymaticregulation now known in biological systems, especially in the regulationof signal transduction from cell surface receptors. Reversiblephosphorylation is important for transmitting regulatory signals,including proliferative ones, in all living cells. To understand themolecular basis of these regulatory mechanisms, it is necessary toidentify the specific amino acid residues that become phosphorylated. Byidentifying the substrates and sites of phosphorylation, diagnostictools may be developed for some tumors and the modification of theprocess itself could be a target for therapeutic intervention.

Polypeptides such as growth factors, differentiation factors andhormones are crucial components of the regulatory system thatcoordinates development of multicellular organisms. Many of thesefactors mediate their pleiotropic actions by binding to and activatingcell surface receptors with an intrinsic protein tyrosine kinaseactivity. Changes in cell behavior induced by extracellular signalingmolecules such as growth factors and cytokines require execution of acomplex program of transcriptional events. To activate or represstranscription, transcription factors must be located in the nucleus,bind DNA, and interact with the basal transcription apparatus.Accordingly, extracellular signals that regulate transcription factoractivity may affect one or more of these processes. Most commonly,regulation is achieved by reversible phosphorylation. Phosphorylation ofa transcription factor by several different kinases (or by a kinaselinked to more than one pathway) is a simple mechanism that allowsdifferent signals to converge at the same factor.

There are a number of approaches in the literature directed to theanalysis of phosphorylation. One such method is two-dimensionalphosphopeptide mapping of ³²P-labeled proteins. Another approach relieson mass spectrometry for analysis of non-radiolabeled phosphoproteins.In another approach (Cao, et al, Rapid Commun. Mass Spectrom. (2000)14:1600-1606) phosphorylation sites of proteins are mapped using on-lineimmobilized metal affinity chromatography (IMAC)/capillaryelectrophoresis (CE)/electrospray ionization multiple stage tandem massspectrometry (MS). The IMAC resin retains and preconcentratesphosphorylated proteins and peptides, CE separates the phosphopeptidesof a mixture eluted from the IMAC resin, and MS provides informationincluding the phosphorylation sites of each component.

A procedure for micropurification of phosphorylated peptides, as a frontend to mass spectrometric analysis, is disclosed by Posewitz, et al.,Anal. Chem. (1999) 71:2883-2892. Immobilized metal affinitychromatography in a microtip format and more specifically, incombination with gallium III ions is employed. Phosphopeptides areretrieved in near quantitative and highly selective manner, to yield aconcentrated sample for direct analysis by matrix-assisted laserdesorption/ionization time of flight and nanoelectrospray ionizationmass spectrometry.

A need still exists, however, for methods for identifying and/ordetermining activity of and/or determining the presence and/or amountsof polypeptides involved in post-translational modification processes.The methods should be able to identify the modification that hasoccurred, the site or sites of modification and the location of thesites of modification. The methods should utilize class-specificreagents where possible and be able to detect multiple polypeptides in asingle assay, i.e., have a high degree of multiplexing capability. Themethods should allow information to be determined in real time and allowa determination of the importance of certain polypeptides in biologicalpathways. Furthermore, it is important that the method permitmultiplexing in order to determine whether a particular pathway isactivated.

SUMMARY OF THE INVENTION

In one aspect the present invention is directed to a method fordetermining the presence and/or amount of one or more targetpolypeptides in a sample suspected of containing the targetpolypeptides. A mixture is formed comprising (i) the sample; (ii) afirst reagent (also referred to herein as a “class-specific reagent”)comprising a cleavage-inducing moiety and a first binding agent specificfor a post-translational modification on one or more targetpolypeptides; and (iii) one or more electrophoretic probes each having abinding moiety specific for a target polypeptide and one or moreelectrophoretic tags each attached thereto by a cleavable linkage. Themixture is subjected to conditions under which binding of respectivebinding agent and moieties occurs. The interaction between the firstbinding agent and the post-translational modification brings thecleavage-inducing moiety into close proximity (also referred to hereinas “effective proximity”) with a cleavable linkage, which is on a probeassociated with the polypeptide and is susceptible to cleavage only whenin proximity to the cleavage-inducing moiety. In this way, uniqueelectrophoretic tags for each of the polypeptides may be released fromthe electrophoretic probe only when binding occurs. The releasedelectrophoretic tags are then separated and the presence and/or amountof the target polypeptides are determined based on the identities andamounts of the corresponding tags. Preferably, each electrophoretic taghas unique optical and/or charge-mass characteristics.

Another embodiment of the present invention is a method of performing amultiplexed assay for the determination of a plurality of targetpolypeptides in a sample where the target polypeptides having undergonephosphorylation. The sample is combined with a first reagent comprisinga cleavage-inducing moiety and a first binding agent comprising anaffinity support and a plurality of electrophoretic probes. Each of theelectrophoretic probes comprises a binding moiety for a respectivetarget polypeptide and a cleavable, or releasable, electrophoretic tag.The combination is subjected to conditions for binding of the firstbinding agent to the target polypeptides. The electrophoretic tag ineach of the electrophoretic probes includes i) a cleavable linkage thatis susceptible to cleavage only when in proximity to a cleavage-inducingmoiety, and ii) a detectable moiety that has unique electrophoreticand/or optical properties. The interaction between the first bindingagent and the target polypeptides brings the cleavage inducing moietyinto close proximity to the cleavable linkage. The electrophoretic tagsare released from the electrophoretic probes, which are bound to thetarget polypeptides, by cleavage of the cleavable linkage. The releasedtags are identified by means of separation and optical characteristicsthat are unique to each tag and the presence of the target polypeptidesin the sample is determined. Preferably, the electrophoretic tags haveunique electrophoretic mobilities and/or fluorescence characteristics.

Another embodiment of the present invention is a composition for use indetecting the presence and/or amount and/or activity of each and any ofa plurality of target polypeptides in a predetermined post-translationalclass, such as phosphorylated proteins, glycoproteins, lipid-derivatizedproteins, or the like. The composition comprises a first reagentcomprising a cleavage-inducing moiety and a first binding agent for abinding site comprising a post-translational modification of a targetpolypeptide. The determination may be for the target polypeptide itselfor an agent involved in the post-translational modification of thetarget polypeptide. The composition may be part of a kit, which alsocomprises in packaged combination a plurality of electrophoretic probeswherein each of the electrophoretic probes comprises a second bindingagent for a respective target polypeptide and a cleavableelectrophoretic tag. The cleavable tag in each of the electrophoreticprobes includes a cleavable moiety that is susceptible to cleavage onlywhen in proximity to a cleavage-inducing moiety, and at least onedetectable moiety having unique electrophoretic and/or opticalcharacteristics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one exemplary synthetic approach starting withcommercially available 6-carboxy fluorescein, where the phenolichydroxyl groups are protected using an anhydride. Upon standardextractive workup, a 95% yield of product is obtained. This material isphosphitylated to generate the phosphoramidite monomer.

FIG. 2 illustrates the use of a symmetrical bis-amino alcohol linker asthe amino alcohol with the second amine then coupled with a multitude ofcarboxylic acid derivatives.

FIG. 3 shows the structure of several benzoic acid derivatives that canserve as mobility modifiers.

FIG. 4 illustrates the use of an alternative strategy that uses5-aminofluorescein as starting material and the same series of steps toconvert it to its protected phosphoramidite monomer.

FIG. 5 illustrates several mobility modifiers that can be used forconversion of amino 5 dyes into e-tag phosphoramidite monomers.

FIGS. 6 A-B illustrate fluorescein derivatives that may be used inconstructing electrophoretic tag of the invention.

FIG. 7 illustrates use of the present invention for proteomic studies.

FIG. 8 is a cartoon illustrating the major phases of the cell cycle andactive molecules and processes in each phase.

FIGS. 9 A-C are cartoon electropherograms illustrating target discoveryand validation using cell-based assays with a-tag reagents and naturalproducts. FIG. 9A shows hypothetical results from an unsynchronized cellpopulation; FIG. 9B shows results from cells arrested in early G1; FIG.9C shows results from cells arrested in late G1.

FIGS. 10 A-F illustrate oxidation-labile linkages and their respectivecleavage reactions mediated by singlet oxygen.

FIG. 11 is a cartoon depicting the use of reagents of the invention todetect the affects of ligand-cell surface receptor interactions.

FIG. 12 is a cartoon with a further depiction of the use of reagents ofthe invention to detect the affects of ligand-cell surface receptorinteractions.

FIG. 13A is a cartoon depicting analysis of protein-protein interactionsin a cellular pathway. FIG. 13B shows hypothetical results of the effectof drug treatments on six designated protein interactions.

FIGS. 14 A-B illustrate the general methodology for conjugation of ane-tag moiety to an antibody to form an a-tag probe, and the reaction ofthe resulting probe with singlet oxygen to produce a sulfinic acidmoiety as the released e-tag reporter.

FIGS. 15 A-J show the structures of e-tag moieties that have beendesigned and synthesized. (Pro1 is commercially available from MolecularProbes, Inc.)

FIGS. 16 A-I illustrate the chemistries of synthesis of the a-tagmoieties illustrated in FIG. 15.

FIGS. 17 A-C are schematic illustrations of a CE² LabCard™ deviceutilized in the present methods. FIG. 17A illustrates the device; FIGS.17B and 17C illustrate exemplary high voltage configurations utilized inthe device for injection and separation, respectively.

FIG. 18 shows two electropherograms demonstrating e-tag reporteranalysis using a CE² LabCard.

FIG. 19 shows multiple electropherograms demonstrating a-tag reporteranalysis using a CE² LabCard.

FIG. 20 depicts the linear calibration curve for the release of e-tagreporters as a function of the photosensitizer bead concentration.

FIG. 21 shows a data curve of the effect of the concentration of labeledaminodextran on a-tag reporter release.

FIG. 22 shows the electrophoretic separation of 8 e-tag reporters on anABI310.

FIG. 23 is a cartoon depicting a sandwich assay for the quantificationof cytokines IL-4 and IL-5.

FIG. 24 shows a series of electropherograms demonstrating e-tag reporter(Pro1) analysis in an IL-4 titration study.

FIG. 25 shows a series of electropherograms demonstrating a-tag reporter(Pro10) analysis in an IL-6 titration study.

FIG. 26 shows a series of electropherograms demonstrating a-tag reporter(Pro8) analysis in an IFN γ titration study.

FIG. 27 shows a series of electropherograms demonstrating a-tag reporter(Pro7) analysis in an TFN α titration study.

FIG. 28 shows a series of electropherograms demonstrating a-tag reporter(Pro4) analysis in an IL-10 titration study.

FIG. 29 shows a series of electropherograms demonstrating a-tag reporter(Pro2) analysis in an IL-8 titration study.

FIG. 30 depicts electropherograms demonstrating a-tag reporter analysisin singleplex and duplex cytokines studies.

FIG. 31 depicts an electropherogram demonstrating a-tag reporteranalysis in a multiplexed study of five cytokines.

FIG. 32 depicts electropherograms demonstrating a-tag reporter analysisin a multiplexed cytokines study.

FIG. 33 is a cartoon depicting a homogeneous assay for the directquantification of human IgG.

FIG. 34 depicts electropherograms demonstrating a-tag reporter analysisin a human IgG titration study.

FIG. 35 depicts a calibration curve quantitating the results of FIG. 34.

DEFINITIONS

As used herein, “alkyldiyl” refers to a saturated or unsaturated,branched, straight-chain or cyclic divalent hydrocarbon radical derivedby the removal of one hydrogen atom from each of two different carbonatoms of a parent alkane, alkene or alkyne, or by the removal of twohydrogen atoms from a single carbon atom of a parent alkane, alkene oralkyne. The two monovalent radical centers or each valency of thedivalent radical center can form bonds with the same or different atoms.Typical alkyldiyls include, but are not limited to, methandiyl;ethyldiyls such as ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl,ethen-1,2-diyl; propyldiyls such as propan-I,I-diyl, propan-1,2-diyl,propan-2,2-diyl, propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan1,2-diyl, prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop en-1,2-diyl,cycloprop en-I,I-diyl, propyn-1,3-diyl, etc.; butyldiyls such as,butan-I,I-diyl, butanyl,2-diyl, butan-I,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-I,I-diyl, 2-methylpropan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-enl,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,17 diyl,2-methanylidene-propan-I,I-diyl, buta-1,3-dien-I,I-diyl,buta-1,3-dien-1,2-diyl, buta1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl, butyn-1,3-diyl, but yn-1,4-diyl, buta-1,3-diyn-1,4-diyl; and the like.

“Antibody” means an immunoglobulin that specifically binds to, and isthereby defined as complementary with, a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera (polyclonal)or by preparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal), or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for specific binding of natural antibodies.Antibodies may include a complete immunoglobulin or fragment thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereofmay include Fab, Fv and F(ab′)2, Fab′, and the like. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular polypeptide is maintained.

“Antibody binding composition” means a molecule or a complex ofmolecules that comprise one or more antibodies and derives its bindingspecificity from an antibody. Antibody binding compositions include, butare not limited to, antibody pairs in which a first antibody bindsspecifically to a target molecule and a second antibody bindsspecifically to a constant region of the first antibody; a biotinylatedantibody that binds specifically to a target molecule and streptavidinderivatized with moieties such as electrophoretic tags orphotosensitizers; antibodies specific for a target molecule andconjugated to a polymer, such as dextran, which, in turn, is derivatizedwith moieties such as electrophoretic tags or photosensitizers;antibodies specific for a target molecule and conjugated to a bead, ormicrobead, or other solid phase support, which, in turn, is derivatizedwith moieties such as electrophoretic tags or photosensitizers, orpolymers containing the latter.

“Capillary electrophoresis” means electrophoresis in a capillary tube orin a capillary plate, where the diameter of the separation column orthickness of the separation plate is between about 25-500 microns,allowing efficient heat dissipation throughout the separation medium,with consequently low thermal convection within the medium.

A “sieving matrix” or “sieving medium” means an electrophoresis mediumthat contains crosslinked or non-crosslinked polymers which areeffective to retard electrophoretic migration of charged species throughthe matrix.

“Specific” in reference to the binding of two molecules or a moleculeand a complex of molecules refers to the specific recognition of one forthe other and the formation of a stable complex as compared tosubstantially less recognition of other molecules and the lack offormation of stable complexes with such other molecules. Preferably,“specific” in reference to binding means that to the extent that amolecule forms complexes with other molecules or complexes, it forms atleast fifty percent of the complexes with the molecule or complex forwhich it has specificity. Generally, the molecules or complexes haveareas on their surfaces or in cavities giving rise to specificrecognition between the two molecules. Exemplary of specific binding areantibody-antigen interactions, enzyme-substrate interactions,polynucleotide interactions, cellular receptor-ligand interactions, andso forth.

As used herein, the term “spectrally resolvable” in reference to aplurality of fluorescent labels means that the fluorescent emissionbands of the labels are sufficiently distinct, i.e. sufficientlynon-overlapping, that electrophoretic tags to which the respectivelabels are attached can be distinguished on the basis of the fluorescentsignal generated by the respective labels by standard photodetectionsystems, e.g. employing a system of band pass filters andphotomultiplier tubes, or the like, as exemplified by the systemsdescribed in U.S. Pat. Nos. 4,230,558, 4,811,218, or the like, or inWheeless et al, pgs. 21-76, in Flow Cytometry: Instrumentation and DataAnalysis (Academic Press, New York, 1985).

DESCRIPTION OF SPECIFIC EMBODIMENTS

In one aspect the present invention is directed to a method fordetermining the presence and/or amount of members of a class of targetpolypeptides in a sample suspected of containing such polypeptides.Classes of protein that are the object of the assays of the inventioninclude proteins having a common physical, functional, or chemicalcharacteristic which provides a means for physical or chemicalidentification. Preferred classes of protein include membrane-boundproteins, proteins having general binding characteristics, such asDNA-binding proteins, and proteins having a specific type ofpost-translational modification, such as phosphorylation, glycosylation,ribosylation, or the like. The preferred classes of target polypeptidesare those polypeptides that have undergone post-translationalmodification.

In another aspect of the invention, it may be desired to determine whatsites on the polypeptides have been modified, how many modifications arepresent on the modified polypeptides, where the modifications are on thepolypeptides, the location of the polypeptides, and so forth. On theother hand, the presence and/or amount of a target polypeptide may beused-to determine the presence and/or amount and/or activity of an agentinvolved in bringing about the post-translational modification of thetarget polypeptide. In this embodiment it is desired to know whether theagent is present and/or active. The agent may be, for example, apolypeptide such as, e.g., an enzyme, a receptor, a complex, e.g., amultimeric protein or a multi-subunit holoenzyme, a protein-nucleicacid, and the like.

Polypeptides are a class of compounds composed of amino acid residueschemically bonded together by amide linkages with elimination of waterbetween the carboxy group of one amino acid and the amino group ofanother amino acid. A polypeptide is a polymer of amino acid residues,which may contain a large number of such residues. Peptides are similarto polypeptides, except that, generally, they are comprised of a lessernumber of amino acids. Peptides are sometimes referred to asoligopeptides. There is no clear-cut distinction between polypeptidesand peptides. For convenience, in this disclosure and claims, the term“polypeptide” will be used to refer generally to peptides andpolypeptides. The amino acid residues may be natural or synthetic.

Proteins are polypeptide chains folded into a defined three-dimensionalstructure. They are complex high polymers containing carbon, hydrogen,nitrogen, and sulfur and are comprised of linear chains of amino acidsconnected by peptide links. The proteins are generally from about 5,000to about 5,000,000 or more in molecular weight, more usually from about5,000 to about 1,000,000 molecular weight. A wide variety of proteinsmay be considered such as a family of proteins having similar structuralfeatures, proteins having particular biological functions, proteinsrelated to specific microorganisms, particularly disease causingmicroorganisms, etc. Such proteins include, by way of illustration andnot limitation, cytokines or interleukins, enzymes such as, e.g.,kinases, proteases, galactosidases and so forth, protamines, histones,albumins, immunoglobulins, scleroproteins, phosphoproteins,mucoproteins, chromoproteins, lipoproteins, nucleoproteins,glycoproteins, T-cell receptors, proteoglycans, unclassified proteins,e.g., somatotropin, prolactin, insulin, pepsin, proteins found in humanplasma, blood clotting factors, blood typing factors, protein hormones,cancer antigens, tissue specific antigens, peptide hormones, nutritionalmarkers, tissue specific antigens, and synthetic peptides.

The preferred focus of the present invention is polypeptides thatinclude amino acid sequences modified by natural processes, such aspost-translational processing. Such modifications are well-described inbasic texts and in more detailed monographs, as well as in a voluminousresearch literature. Modifications may occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present to the same or varying degrees at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications. Polypeptides may be branched as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched and branched cyclic polypeptides may result frompost-translational natural processes. It is also within the purview ofthe present invention that the modification is the result of anon-natural activity such as chemical modification.

Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance,Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton,W.H. Freeman and Company, New York, 1993; Wold, F., Post-translationalProtein Modifications: Perspectives and Prospects, pgs. 1-12 inPost-translational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press, New York, 1983; Seifter et al., “Analysis forprotein modifications and non-protein cofactors”, Meth Enzymol (1990)182:626-646 and Rattan et al., “Protein Synthesis: Post-translationalModifications and Aging”, Ann NY Acad Sci (1992) 663:48-62).

The sample for determining post-translational modification activity isusually material derived from cells. The sample can be obtained by lysisof cells, from serum, plasma, saliva, blood, or other bodily fluid. Abiological pathway may be analyzed in buffer, e.g., detection ofpost-translational modification of a receptor by an enzyme or anotherreceptor.

Class-Specific Reagent

One reagent for conducting methods in accordance with the presentinvention is a class-specific reagent, or generic reagent, thatcomprises a cleavage-inducing moiety and a binding agent for a bindingsite on all, or substantially all, members of a class of targetpolypeptide. This reagent is a generic in the sense that its bindingagent binds to all or nearly all members of a particular class ofproteins. Preferably, the binding agent of the class-specific reagent isselected so that unbound materials can be separated easily from thebound material if desired.

In one embodiment, immobilized metal affinity chromatography (IMAC) isused to capture on a solid phase, such as beads, all phosphorylatedproteins contained in a sample, such as a cell lysate, e.g. as disclosedin Holmes, J. Liquid Chromatography and R^(e1). Technol., 20: 123-142(1997); Posewitz et al, Anal. Chem., 71: 2883-2892 (1999); or the like.Following binding, the beads are washed by filtration. The capture andwash steps serve to concentrate the phosphoproteins and removecontaminating non-phosphorylated proteins and other cellular debris fromthe assay. The proteins bound to the beads are resuspended in a solutioncontaining antibody to the candidate protein(s) of interest. Antibodymay be specific for one or more designated protein targets of interest,or may be a polyclonal antibody reagent prepared against whole-celllysate. Preferably, a collection of monoclonal antibodies is employedwherein one or more different monoclonal antibodies are specific foreach phosphorylated protein in a predetermined set of such proteins. Theantibody reagent has one or more e-tag moieties cleavably-linkedthereon, where the linkage is susceptible to cleavage by acleavage-inducing moiety contained on an IMAC resin, such as IMAC-24DNP. Multiple antibody reagents, each specific for a differentdesignated protein and each linked to a designated a-tag moiety uniquelyassigned to the designated protein, may be combined for a multiplexed,target-specific determination. Following antibody binding, the linkageto the a-tag moieties will be cleaved to release a corresponding a-tagreporter, indicating capture of the designated target by the IMAC-24 DNPbead. The protein-specific or epitope-specific monoclonal antibodies mayhave e-tag moieties attached directly, or the a-tags may be attached toa secondary antibody specific for a constant region of the monoclonalantibody bound to a selected protein.

The binding site on the polypeptide is usually the result of thepost-translational modification of a polypeptide. Accordingly, thebinding site may be any one of the modifications mentioned above. Thebinding agent for the binding site on the polypeptide is, therefore,dependent on the nature of the binding site or modification. Usually,the binding agent is an affinity reagent that is capable of specificrecognition of the modification. The following table (Table 1a) setsforth various post-translational modifications and corresponding bindingagents:

TABLE 1a Modification Binding Agent Phosphorylation Metal affinityagent + metal Antibodies Biotin Covalent modification of —O—PO₃ ⁼Glycosylation Boronic acid-containing agents Lectins AntibodiesLipidation Antibodies Cyclodeschins Lectins Formation of disulfidebridges Antibodies Nitrotyrosine Antibodies Ubiquitination Antibodies

Metal Affinity Agent

In one embodiment of the invention a metal affinity agent in combinationwith an appropriate metal may be employed as the binding agent. Themetal affinity agent is one that is designed to chelate a certain metalion that has selectivity for specific groups. Accordingly, any ligandhaving affinity for a metal ion that binds to a binding site resultingfrom post-translational modification may be employed. Thus, the natureof the chelating ligand is dependent on the metal ion, which in turn isdependent on the post-translational modification. The term “metal ion”refers to ions that are derived from, for example, simple salts (e.g.,AiCl3, NiCl2, etc.), complex or mixed salts comprising both organic andinorganic ligands and metal complexes. Metal ions of use in practicingthe present invention include, for example, main group metal ions,transition metal ions, lanthanide ions, etc. Zero valent metalprecursors are included in this definition. Examples of such metal ionsinclude, by way of illustration and not limitation, ions of gallium,aluminum, iron, lead, mercury, nickel, cadmium, thallium, antimony,silver, chromium, manganese, platinum, gold, bismuth, iron, copper,zinc, cobalt, molybdenum, selenium, vanadium, calcium, Eu, Gd, Tb, Sm,and so forth.

For phosphate-containing moieties such as those arising fromphosphorylation of polypeptides, suitable metal ions include thosehaving a valency of 2 or 3. Particularly preferred metal ions aregallium III, aluminum III, iron III, CO⁺³, EU⁺³, Gd⁺³, SM⁺³, Tb⁺³.

The chelating ligand is usually bidentate, tridentate, or quadradentatein that the chelating ligand comprises about 2 to about 4 metalcoordinating sites. The coordinating sites my comprise nitrogen, such asimino, nitrilo, pyridinyl, pyrazolyl, imidazolyl, isocyanidyl, and soforth; oxygen, such as carboxy, hydroxy, ether, keto, and so forth;phosphorus, such as phosphine, and so forth; arsenic, such as arsine,and so forth; antimony, such as stilbines, and so forth; sulfur, such asthioether, thioketo, and so forth; selenium, such as selenoether, and soforth; tellurium, such as teluroether, and so forth; and the like. Alsoincluded are combinations of the aforementioned, such as, for example,thiocarboxy, phosphinimino, oxazoles, oxazolines, thiophenes, thiazoles,isoxazoles, isothrazoles, and the like. Also included are organicmoieties such as arenes, acetylenes, olefins, and the like. Specificexamples of chelating ligands comprising the aforementioned groupsinclude, by way of illustration and not limitation, iminodiacetate,tris(carboxymethyl)ethylenediamine, nitrilotriacetic acid usuallysubstituted in the alpha position by alkyl (1-30 carbon atoms),carboxymethylated aspartic acid, 2-hydroxy 3[N-(2-pyridylmethyl)glycine]propyl, and the like.

The chelating ligand may be a metal binding peptide such as, forexample, (GHHPH)_(n)G wherein n is 1 (SEQ ID NO:1), 2 (SEQ ID NO:2), 3(SEQ ID NO:3) or 5 (SEQ ID NO:4)) (see, for example, Hutchens, et al.,J. Chromatogr. (1992) 604:125-132 and 133-141), and so forth.

Many of the aforementioned metal chelating ligands are commerciallyavailable, others have been synthesized and the synthesis is part of theliterature. Other metal chelating ligands may be synthesized byprocedures well known in the art.

Boronic Acid-Containing Agent

In one embodiment of the invention the binding agent is a boronic acidmoiety, which includes at least one boron atom substituted with moietiesthat permit complex formation with the interactive functionalities ofthe binding site on the polypeptide. Usually, the boronic acid moiety isderived from boronic acid that is substituted with an organic moietyhaving at least about 2 atoms selected from the group consisting ofcarbon, oxygen, nitrogen, sulfur, and phosphate. Usually, the organicmoiety has at least about 2 carbon atoms which may be substituted orunsubstituted. The organic moiety may be aliphatic or aromatic. Animportant consideration regarding the boronic acid moiety is itsacidity. In general, the higher the acidity of the boronic acid moiety,the better is its ability to complex with the interactivefunctionalities of the binding site. Desirably, the pKa of the boronicacid moiety is below about 11, preferably below about 9, morepreferably, below about 8.75. The lower the pKa of the boronic acidmoiety, the better the ability to bind to the binding site of thepolypeptide. Accordingly, substituents on the boron that enhance theacidity over that of boronic acid are preferred. Aromatic substituentson the boron are preferred such as, for example, phenyl and substitutedphenyl (substituted with one or more functionalities such as amino,nitro, and the like. To enhance the acidity of the boronic acid moiety,the aromatic substituents preferably contain one or moreelectron-withdrawing groups such as, for example, nitro, and the like.Specific examples of organic moieties for the boronic acid moietyinclude phenyl, aminophenyl, and so forth. Specific boronic acidmoieties include, by way of illustration and not limitation, phenylboronic acid and (3-aminophenyl)boronic acid. Other examples may befound in U.S. Pat. Nos. 5,623,055, 5,876,938, 6,013,783, 5,831,045, therelevant disclosures of which are incorporated herein by reference.

Many of the aforementioned boronic acid containing agents arecommercially available, others have been synthesized and the synthesisis part of the literature. Other metal boronic acid containing agentsmay be synthesized by procedures well known in the art.

Lectin Agent

In another embodiment of the invention a lectin may be employed as thebinding agent. Lectins are proteins or glycoproteins that have receptorsite specificity for a particular sugar or sugars but not for othersugars. Accordingly, lectins may be used as binding agents for detectionof glycosylation. For example, Concanavalian A (Con A) has specificityfor alpha-D glucose and alpha-D-mannose. When a ligand such as glucoseis present on a polypeptide, Con A binds to the glucosylatedpolypeptide. The lectins may be from any suitable source such as, forexample, plant, mammal, microorganism, and so forth. The number of knownlectins is too numerous to list here. As indicated above, the lectinsare specific for a particular sugar or sugars. Accordingly, the lectinis chosen based on the expected glycosylation moiety for thepolypeptide. Examples of lectins, by way of illustration and notlimitation, include, Concanavalian A, agglutinins such as, e.g., wheatgerm agglutinin, Sambucus nigra agglutinin (SNA), Arachis HypogaeaAgglutinin, Bauhinia Purpurea Agglutinin, Galanthus nivalis agglutinin(GNA), Datura stramionium agglutinin (DSA), Maackia amurensis agglutinin(MAA), peanut agglutinin etc., elderberry bark lectin, Ulex Europeus(UEA I), Ulex Europaeus (UEA II), Limulus Polyhemus (LPA), LotusTetragonolobus (Lotus A), and so forth.

Antibody Agent

In one embodiment the binding agent may be an antibody for themodification on the polypeptide. For example, antibodies that recognizephosphate groups may be employed for phosphorylated polypeptides, orantibodies that recognize a sugar moiety may be employed forglucosylated polypeptides, or antibodies that recognize acetylation maybe employed for acetylated polypeptides, and so forth. The antibody canbe monoclonal or polyclonal. Many suitable antibodies are known in theart and/or can be prepared by techniques that are well known in the art.Such techniques include immunization of a host and collection of sera(polyclonal), by preparing continuous hybrid cell lines and collectingthe secreted protein (monoclonal) or by cloning and expressingnucleotide sequences or mutagenized versions thereof coding at least forthe amino acid sequences required for specific binding of naturalantibodies. Antibodies include complete immunoglobulins or fragmentsthereof, which immunoglobulins include the various classes and isotypes,such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b, and IgG3, IgM, etc. Fragmentsthereof may include Fab, Fv and F(ab′)2 Gab′, and the like. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular molecule is maintained.

In one approach to the preparation of a suitable antibody, antiserumcontaining antibodies (polyclonal) is obtained by well-establishedtechniques involving immunization of an animal, such as a rabbit, guineapig, or goat, with an appropriate immunogen and obtaining antisera fromthe blood of the immunized animal after an appropriate waiting period.State-of-the-art reviews are provided by Parker, Radioimmunoassay ofBiologically Active Compounds, Prentice-Hall (Englewood Cliffs, N.J.,U.S., 1976), Butler, J. Immunol. Meth. 7: 1-24 (1975); Broughton andStrong, Clin. Chem. 22: 726-732 (1976); and Playfair, et al., Br. Med.Bull. 30: 24-31 (1974). Antibodies can also be obtained by somatic cellhybridization techniques, such antibodies being commonly referred to asmonoclonal antibodies. Monoclonal antibodies may be produced accordingto the standard techniques of Kohler and Milstein, Nature 265:495-497,1975. Reviews of monoclonal antibody techniques are found in LymphocyteHybridomas, ed. Melchers, et al. Springer-Verlag (New York 1978), Nature266: 495 (1977), Science 208: 692 (1980), and Methods of Enzymology 73(Part B): 3-46 (1981). Various techniques exist for enhancing yields ofmonoclonal antibodies, such as injection of the hybridoma cells into theperitoneal cavity of a mammalian host, which accepts the cells, andharvesting the ascites fluid. Where an insufficient amount of themonoclonal antibody collects in the ascites fluid, the antibody isharvested from the blood of the host. Alternatively, the cell producingthe desired antibody can be grown in a hollow fiber cell culture deviceor a spinner flask device, both of which are well known in the art.Various conventional ways exist for isolation and purification of themonoclonal antibodies from other proteins and other contaminants (seeKohler and Milstein, supra). In another approach for the preparation ofantibodies the sequence coding for antibody binding sites can be excisedfrom the chromosome DNA and inserted into a cloning vector which can beexpressed in bacteria to produce recombinant proteins having thecorresponding antibody binding sites. In general, antibodies can bepurified by known techniques such as chromatography, e.g., DEAEchromatography, ABx chromatography, and the like, filtration, and soforth.

Biotin Agent

In one embodiment, phospho groups of phosphoproteins may be biotinylatedand the proteins isolated by streptavidin, Goshe et al, Anal. Chem., 73:2578 (2001).

Cleavage-Inducing Moiety

A cleavage-inducing moiety is a group that produces an active speciesthat is capable of cleaving a cleavable linkage, preferably byoxidation. Preferably, the active species is a chemical species thatexhibits short-lived activity so that its cleavage-inducing effects areonly in the proximity of the site of its generation. Either the activespecies is inherently short lived, so that it will not createsignificant background because beyond the proximity of its creation, ora scavenger is employed that efficiently scavenges the active species,so that it is not available to react with cleavable linkages beyond ashort distance from the site of its generation. Illustrative activespecies include singlet oxygen, hydrogen peroxide, NADH, and hydroxylradicals, phenoxy radical, superoxide, and the like. Illustrativequenchers for active species that cause oxidation include polyenes,carotenoids, vitamin E, vitamin C, amino acid-pyrrole N-conjugates oftyrosine, histidine, and glutathione, and the like, e.g. Beutner et al,Meth. Enzymol., 319: 226-241 (2000).

An important consideration for the cleavage-inducing moiety and thecleavable linkage is that they not be so far removed from one anotherwhen bound to a target protein that the active species generated by thesensitizer diffuses and loses its activity before it can interact withthe cleavable linkage. Accordingly, a cleavable linkage preferably arewithin 1000 nm, preferably 20-100 nm of a bound cleavage-inducingmoiety. This effective range of a cleavage-inducing moiety is referredto herein as its “effective proximity.”

Generators of active species include enzymes, such as oxidases, such asglucose oxidase, xanthene oxidase, D-amino acid oxidase, NADH-FMNoxidoreductase, galactose oxidase, glyceryl phosphate oxidase, sarcosineoxidase, choline oxidase and alcohol oxidase, that produce hydrogenperoxide, horse radish peroxidase, that produces hydroxyl radical,various dehydrogenases that produce NADH or NADPH, urease that producesammonia to create a high local pH. One cleavable linkage can be based onthe oxidation of sulfur or selenium, where a thioether, sulfoxide, orselenium analog thereof, is present at the α- or β-position in relationto an activating group, which makes the hydrogen a to the activatinggroup acidic and capable of being removed by base, so as to release theoxidized functionality to which is attached the releasable portion ofthe e-tag, or to be subject to oxidation with release of the e-tag.Alternatively, one may use metal chelates that are stable at oneoxidation state and unstable at another oxidation state. Other compoundsinclude α-substituted methylquinones, which have the releasable portionof a reagent bonded through a leaving group, such as sulfonyl, oxy,amino, etc.

A sensitizer is a molecule, usually a compound, that can be induced togenerate a reactive intermediate, or species, usually singlet oxygen.Preferably, a sensitizer used in accordance with the invention is aphotosensitizer. However, other sensitizers can be employed in thepresent invention such as, for example, chemi-activated (e.g., enzymesand metal salts) including, by way of example and not limitation, othersubstances and compositions that can produce singlet oxygen with or,less preferably, without activation by an external light source. Thus,for example, molybdate (MoO₄ ⁼) salts and chloroperoxidase andmyeloperoxidase plus bromide or chloride ion have been shown to catalyzethe conversion of hydrogen peroxide to singlet oxygen and water. For theabove examples of sensitizers, hydrogen peroxide may be included as anancillary reagent, chloroperoxidase may be bound to a surface andmolybdate may be incorporated in the aqueous phase of a liposome,respectively. Other sensitizers included within the scope of theinvention are compounds that are not true sensitizers but which onexcitation by heat, light, ionizing radiation, or chemical activationwill release a molecule of singlet oxygen. The best known members ofthis class of compounds include the endoperoxides such as1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen. Further sensitizers aredisclosed in the following references: Di Mascio et al, FEBS Lett., 355:287 (1994)(peroxidases and oxygenases); Kanofsky, J. Biol. Chem. 258:5991-5993 (1983)(lactoperoxidase); Pierlot et al, Meth. Enzymol., 319:3-20 (2000)(thermal lysis of endoperoxides); and the like.

Attachment of a binding agent to the cleavage-inducing moiety may bedirect or indirect, covalent or non-covalent and can be accomplished bywell-known techniques, commonly available in the literature. See, forexample, “Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York(1978); Cuatrecasas, J. Biol. Chem., 245:3059 (1970). A wide variety offunctional groups are available or can be incorporated. Functionalgroups include carboxylic acids, aldehydes, amino groups, cyano groups,ethylene groups, hydroxyl groups, mercapto groups, and the like. Themanner of linking a wide variety of compounds is well known and is amplyillustrated in the literature (see above). The length of a linking groupto a binding agent may vary widely, depending upon the nature of thecompound being linked, the effect of the distance on the specificbinding properties and the like.

Lectins may be attached to the cleavage-inducing moiety by knowncovalent bonding techniques. Such binding is suitably performed bycross-linking the lectin with the cleavage-inducing moiety or a hubmolecule through a bifunctional cross-linking agent. Suitablebifunctional compounds are found in Review by Peters, K. and Richards,F. M., (Ann. Rev. Biochim. 46 (1977) 523). Alkyl imidates show a highdegree of specificity among the functional groups presented to them by aprotein. The reaction is specific for primary amino groups. Specificcoupling reagents include amidoesters such as dimethyl malonimidate,azides such as the acryl azide of tartryl diazide which reacts readilywith amino groups to produce amidelinkages, aryl dihalides (e.g.,1,5-difluoro-2,4-dinitrobenzene, or 4,4′-difluoro-3,3′-dinitrophenylsulfone), glutaraldehyde, dimaleimide, mixed anhydride, mixed aromaticor aliphatic dicarboxyl, N-hydroxysuccimide ester, and other knowncross-linking agents. Catalytic reagents such as1-ethyl-3(3-dimethylamino propyl)carbodiimide hydrochloride may be usedto form covalent bonds between amino groups of one molecule to carboxylgroups of another.

The class-specific reagent may be preformed or formed in situ. In theformer circumstance the class-specific reagent has all of its componentsbound together prior to use in the present methods. In the lattersituation at least some of the components of the class-specific reagentare added separately to a medium in which the present methods areconducted. In one approach the binding agent comprises a moiety forattachment of the cleavage-inducing moiety. Usually, this involves asecond moiety, which is present on the cleavage-inducing moiety, wherethe second moiety and the moiety of the binding agent interact providingfor attachment of the cleavage-inducing moiety to the binding agent andformation of the class-specific reagent in situ. Typically, the moietiesinteract by non-covalent attachment. This situation is exemplified byone of the two moieties comprising a small molecule (about 100 to about1500 molecular weight) and the other of the moieties comprising abinding partner for the small molecule. For example, the small moleculemay be biotin, digoxin, fluorescein, dinitrophenol, and so forth, andthe binding partner for the small molecule is, respectively, avidin,antibody for digoxin, antibody for fluorescein, antibody fordinitrophenol, and so forth.

It may be desirable to have multiple cleavage-inducing moieties attachedto a binding agent to increase, for example, the number of activespecies generated. In one approach the binding agent has a plurality ofsites for attachment such as, for example, an antibody, a lectin, and soforth. To further enhance the number of cleavage-inducing moieties, ahub molecule or nucleus is employed. The hub nucleus is a polyfunctionalmaterial, normally polymeric, having a plurality of functional groups,e.g., hydroxy, amino, mercapto, carboxy, ethylenic, aldehyde, etc., assites for linking. The functionalities on the hub should be those thatare reactive with a functionality on the cleavage-inducing moiety or thebinding agent to be attached. A discussion of hub nuclei is set forthbelow with respect to other reagents and the principles discussed belowmay be applied in this instance as well.

In certain embodiments the class-specific reagent comprises a supportwith which one of the components of the class-specific reagent isassociated. The support may be comprised of an organic or inorganic,solid or fluid, water insoluble material, which may be transparent orpartially transparent. The support can have any of a number of shapes,such as particle including bead, film, membrane, tube, well, strip, rod,and the like. For supports in which photosensitizer is incorporated, thesurface of the support is, preferably, hydrophilic or capable of beingrendered hydrophilic and the body of the support is, preferably,hydrophobic. The support may be suspendable in the medium in which it isemployed. Examples of suspendable supports, by way of illustration andnot limitation, are polymeric materials such as latex, lipid bilayers,oil droplets, cells and hydrogels. Other support compositions includeglass, metals, polymers, such as nitrocellulose, cellulose acetate,poly(vinyl chloride), polyacrylamide, polyacrylate, polyethylene,polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate,poly(ethylene terephthalate), nylon, poly(vinyl butyrate), etc.; eitherused by themselves or in conjunction with other materials. Attachment ofbinding agents to the support may be direct or indirect, covalent ornon-covalent and can be accomplished by well-known techniques, commonlyavailable in the literature as discussed above. See, for example,“Immobilized Enzymes,” Ichiro Chibata, supra. The surface of the supportwill usually be polyfunctional or be capable of being polyfunctionalizedor be capable of binding to a target-binding moiety, or the like,through covalent or specific or non-specific non-covalent interactions.

The cleavage-inducing moiety may be associated with the support by beingcovalently or non-covalently attached to the surface of the support orincorporated into the body of the support. Linking to the surface may beaccomplished as discussed above. The cleavage-inducing moiety may beincorporated into the body of the support either during or after thepreparation of the support. In general, the cleavage-inducing moiety isassociated with the support in an amount necessary to achieve thenecessary amount of active species. Generally, the amount of cleavageinducing moiety is determined empirically.

Photosensitizers as Cleavage-Inducing Moieties

As mentioned above, the preferred cleavage-inducing moiety in accordancewith the present invention is a photosensitizer that produces singletoxygen. As used herein, “photosensitizer” refers to a light-adsorbingmolecule that when activated by light converts molecular oxygen intosinglet oxygen. Photosensitizers may be attached directly or indirectly,via covalent or non-covalent linkages, to the binding agent of aclass-specific reagent. Guidance for constructing of such compositions,particularly for antibodies as binding agents, available in theliterature, e.g. in the fields of photodynamic therapy,immunodiagnostics, and the like. The following are exemplary references:Ullman, et al., Proc. Natl. Acad. Sci. USA 91, 5426-5430 (1994); Stronget al, Ann. New York Acad. Sci., 745: 297-320 (1994); Yarmush et al,Crit. Rev. Therapeutic Drug Carrier Syst., 10: 197-252 (1993); Pease etal, U.S. Pat. No. 5,709,994; Ullman et al, U.S. Pat. No. 5,340,716;Ullman et al, U.S. Pat. No. 6,251,581; McCapra, U.S. Pat. No. 5,516,636;and the like.

Likewise, there is guidance in the literature regarding the propertiesand selection of photosensitizers suitable for use in the presentinvention. The following are exemplary references: Wasserman and R. W.Murray. Singlet Oxygen. (Academic Press, New York, 1979); Baumstark,Singlet Oxygen, Vol. 2 (CRC Press Inc., Boca Raton, Fla. 1983); andTurro, Modern Molecular Photochemistry (University Science Books, 1991).

The photosensitizers are sensitizers for generation of singlet oxygen byexcitation with light. The photosensitizers include dyes and aromaticcompounds, and are usually compounds comprised of covalently bondedatoms, usually with multiple conjugated double or triple bonds. Thecompounds typically absorb light in the wavelength range of about 200 toabout 1,100 nm, usually, about 300 to about 1,000 nm, preferably, about450 to about 950 nm, with an extinction coefficient at its absorbancemaximum greater than about 500 M⁻¹ cm⁻¹, preferably, about 5,000 M⁻¹cm⁻¹, more preferably, about 50,000 M⁻¹ cm⁻¹, at the excitationwavelength. The lifetime of an excited state produced followingabsorption of light in the absence of oxygen will usually be at leastabout 100 nanoseconds, preferably, at least about 1 millisecond. Ingeneral, the lifetime must be sufficiently long to permit cleavage of alinkage in a reagent in accordance with the present invention. Such areagent is normally present at concentrations as discussed below. Thephotosensitizer excited state usually has a different spin quantumnumber (S) than its ground state and is usually a triplet (S=1) when theground state, as is usually the case, is a singlet (S=0). Preferably,the photosensitizer has a high intersystem crossing yield. That is,photoexcitation of a photosensitizer usually produces a triplet statewith an efficiency of at least about 10%, desirably at least about 40%,preferably greater than about 80%.

Photosensitizers chosen are relatively photostable and, preferably, donot react efficiently with singlet oxygen. Several structural featuresare present in most useful photosensitizers. Most photosensitizers haveat least one and frequently three or more conjugated double or triplebonds held in a rigid, frequently aromatic structure. They willfrequently contain at least one group that accelerates intersystemcrossing such as a carbonyl or mine group or a heavy atom selected fromrows 3-6 of the periodic table, especially iodine or bromine, or theymay have extended aromatic structures.

A large variety of light sources are available to photo-activatephotosensitizers to generate singlet oxygen. Both polychromatic andmonchromatic sources may be used as long as the source is sufficientlyintense to produce enough singlet oxygen in a practical time duration.The length of the irradiation is dependent on the nature of thephotosensitizer, the nature of the cleavable linkage, the power of thesource of irradiation, and its distance from the sample, and so forth.In general, the period for irradiation may be less than about amicrosecond to as long as about 10 minutes, usually in the range ofabout one millisecond to about 60 seconds. The intensity and length ofirradiation should be sufficient to excite at least about 0.1% of thephotosensitizer molecules, usually at least about 30% of thephotosensitizer molecules and preferably, substantially all of thephotosensitizer molecules. Exemplary light sources include, by way ofillustration and not limitation, lasers such as, e.g., helium-neonlasers, argon lasers, YAG lasers, He/Cd lasers, and ruby lasers;photodiodes; mercury, sodium and xenon vapor lamps; incandescent lampssuch as, e.g., tungsten and tungsten/halogen; flashlamps; and the like.

Examples of photosensitizers that may be utilized in the presentinvention are those that have the above properties and are enumerated inthe following references: Turro, Modern Molecular Photochemistry (citedabove); Singh and Ullman, U.S. Pat. No. 5,536,834; Li et al, U.S. Pat.No. 5,763,602; Ullman, et al., Proc. Natl. Acad. Sci. USA 91, 5426-5430(1994); Strong et al, Ann. New York Acad. Sci., 745: 297-320 (1994);Martin et al, Methods Enzymol., 186: 635-645 (1990); Yarmush et al,Crit. Rev. Therapeutic Drug Carrier Syst., 10: 197-252 (1993); Pease etal, U.S. Pat. No. 5,709,994; Ullman et al, U.S. Pat. No. 5,340,716;Ullman et al, U.S. Pat. No. 6,251,581; McCapra, U.S. Pat. No. 5,516,636;Wohrle, Chimia, 45: 307-310 (1991); Thetford, European patent publ.0484027; Sessler et al, SPIE, 1426: 318-329 (1991); Madison et al, BrainResearch, 522: 90-98 (1990); Polo et al, Inorganica Chimica Acta, 192:1-3 (1992); Demas et al, J. Macromol. Sci., A25: 1189-1214 (1988); andthe like. Exemplary photosensitizers are listed in Table 1b.

TABLE 1b Exemplary Photosensitizers Hypocrellin A TetraphenylporphyrinHypocrellin B Halogenated derivatives of rhodamine dyes Hypericinmetallo-Porphyrins Halogenated derivatives of Phthalocyaninesfluorescein dyes Rose bengal Naphthalocyanines Merocyanine 540Texaphyrin-type macrocycles Methylene blue Hematophorphyrin9-Thioxanthone 9,10-Dibromoanthracene Chlorophylls BenzophenonePhenaleone Chlorin e6 Protoporphyrin Perylene Benzoporphryin A monacidBenzoporphryin B monacid

In certain embodiments the photosensitizer moiety comprises a support,as discussed above with respect to the cleavage-inducing moiety. Thephotosensitizer may be associated with the support by being covalentlyor non-covalently attached to the surface of the support or incorporatedinto the body of the support as discussed above. In general, thephotosensitizer is associated with the support in an amount necessary toachieve the necessary amount of singlet oxygen. Generally, the amount ofphotosensitizer is determined empirically. Photosensitizers used as thephotosensitizer are preferably relatively non-polar to assuredissolution into a lipophilic member when the photosensitizer isincorporated in, for example, a latex particle to form photosensitizerbeads, e.g. as disclosed by Pease et al., U.S. Pat. No. 5,709,994. Forexample, the photosensitizer rose bengal is covalently attached to 0.5micron latex beads by means of chloromethyl groups on the latex toprovide an ester linking group, as described in J. Amer. Chem. Soc., 97:3741 (1975).

In one aspect of the invention, a class-specific reagent comprises afirst binding agent that is an antibody and a cleavage-inducing moietythat is a photosensitizer, such that the photosensitizer is covalentlylinked to the antibody, e.g. using well know techniques as disclosed inStrong et al (cited above); Yarmush et al (cited above); or the like.Alternatively, a class specific reagent comprises a solid phase support,e.g. a bead, to which a photosensitizer is covalently or non-covalentlyattached and an antibody is attached, preferably convalently, eitherdirectly or by way of a functionalized polymer, such as amino-dextran,or the like.

Electrophoretic Probe Compositions

According to an important feature of the invention, there is provided aset of electrophoretic probes, each of which has a uniquepolypeptide-binding moiety and an associated “e-tag moiety” that has aunique charge to mass ratio and/or optical characteristic. Forconvenience, the unique charge to mass ratio of an e-tag moiety is dueto the chemical structure of the mobility modifier, since the detectiongroup and linking-group residue (if any) will be common to any set ofelectrophoretic probes. However, it is recognized that unique chargeand/or mass contributions to the e-tag reporters can be made by thedetection group as well. For example, a set of electrophoretic probesmaybe made up of a first subset having a group of mobility modifierswhich impart unique electrophoretic mobilities to the subset incombination with a detection group having one defined charge and/ormass, and a second subset having the same group of mobility modifiers incombination with a second detection group with a different charge and/ormass, thus to impart electrophoretic mobilities which are unique amongboth subsets.

In one aspect, the invention includes compositions comprisingpluralities of electrophoretic probes. An electrophoretic probecomprises a binding moiety specific for a target polypeptide and one ormore electrophoretic tags. The electrophoretic tags may be attached tothe binding moiety directly or indirectly by a secondary bindingmolecule that binds specifically to the binding moiety, such as asecondary antibody specific for the constant region of a primaryantibody. Preferably, the binding moiety of an electrophoretic probe isan antibody. Generally, an electrophoretic probe is defined by thefollowing formula:T-(L-E)_(k)wherein T is the binding moiety, or more specifically, apolypeptide-binding moiety; L is a cleavable linkage; and E is anelectrophoretic tag, or “e-tag.” Preferably, cleavable linkage, L, is anoxidation-labile linkage, and more preferably, it is a linkage that maybe cleaved by singlet oxygen. The moiety “-(L-E)_(k)” indicates that asingle binding moiety may have one or more electrophoretic tags attachedvia cleavable linkages. k is an integer greater than or equal to 1; andpreferably, k is an integer in the range of from 1 to 500; and morepreferably, k is an integer in the range of from 1 to 100 or from 1 to50; and still more preferably, k is an integer in the range of from 1 to10. Preferably, the plurality of electrophoretic probes is at least 5,and more preferably, at least 10. Still more preferably, the pluralityis in the range of from 5 to 200, and more preferably, from 5 to 100, orfrom 5 to 50, or from 10 to 30. Preferably, within a plurality, eachdifferent binding moiety, T, has a different electrophoretic tag, E.Oxidation-labile linkages and tags, E, are attached to T by way ofconventional linking chemistries. Preferably, whenever T is apolypeptide attachment may be through the common reactivefunctionalities, such as amino, sulfide, carboxyl, and the like.

Preferably, binding moiety, T, is an antibody, or comprises an antibody,specific for a target protein, or polypeptide. In the latter case, T maycomprise a plurality of binding components that operate together to holdan electrophoretic tag in the proximity of a target protein. Forexample, T may be an antibody together with a secondary antibody havinge-tags attached, a haptenized antibody together with a secondaryanti-hapten antibody having e-tags attached, a biotinylated antibodytogether with streptavidin having a-tags attached, an antibodyderivatized with a functionalized polymer that, in turn, has a-tagsattached, or the like. A plurality of electrophoretic probes arepreferably used in the method of the invention, wherein each probe has adifferent binding moiety, T.

Preferably, L is a thioether or its selenium analog; or an olefin, whichcontains carbon carbon double bonds, wherein cleavage of a double bondto an oxo group, releases the e-tag, E. Illustrative olefins includevinyl sulfides, vinyl ethers, enamines, imines substituted at the carbonatoms with an a-methine (CH, a carbon atom having at least one hydrogenatom), where the vinyl group may be in a ring, the heteroatom may be ina ring, or substituted on the cyclic olefinic carbon atom, and therewill be at least one and up to four heteroatoms bonded to the olefiniccarbon atoms. The resulting dioxetane may decompose spontaneously, byheating above ambient temperature, usually below about 75° C., byreaction with acid or base, or by photo-activation in the absence orpresence of a photosensitizer. Such reactions are described in thefollowing exemplary references: Adam and Liu, J. Amer. Chem. Soc. 94,1206-1209, 1972, Ando, et al., J. C. S. Chem. Comm. 1972, 477-8, Ando,et al., Tetrahedron 29, 1507-13, 1973, Ando, et al., J. Amer. Chem. Soc.96, 6766-8, 1974, Ando and Migita, ibid. 97, 5028-9, 1975, Wasserman andTerao, Tetra. Lett. 21, 1735-38, 1975, Ando and Watanabe, ibid. 47,4127-30, 1975, Zaklika, et al., Photochemistry and Photobiology 30,35-44, 1979, and Adam, et al., Tetra. Lett. 36, 7853-4, 1995. See also,U.S. Pat. No. 5,756,726.

The formation of dioxetanes is obtained by the reaction of singletoxygen with an activated olefin substituted with an e-tag moiety at onecarbon atom and the binding moiety at the other carbon atom of theolefin. See, for example, U.S. Pat. No. 5,807,675. These cleavablelinkages may be depicted by the following formula:—W—(X)_(n)C_(α)═C_(β)(Y)(Z)—wherein:

W may be a bond, a heteroatom, e.g., O, S, N, P, M (intending a metalthat forms a stable covalent bond), or a functionality, such ascarbonyl, imino, etc., and may be bonded to X or C_(a); at least one Xwill be aliphatic, aromatic, alicyclic or heterocyclic and bonded toC_(α) through a hetero atom, e.g., N, O, or S and the other X may be thesame or different and may in addition be hydrogen, aliphatic, aromatic,alicyclic or heterocyclic, usually being aromatic or aromaticheterocyclic wherein one X may be taken together with Y to form a ring,usually a heterocyclic ring, with the carbon atoms to which they areattached, generally when other than hydrogen being from about 1 to 20,usually 1 to 12, more usually 1 to 8 carbon atoms and one X will have 0to 6, usually 0 to 4 heteroatoms, while the other X will have at leastone heteroatom and up to 6 heteroatoms, usually 1 to 4 heteroatoms;

Y will come within the definition of X, usually being bonded to C_(β)through a heteroatom and as indicated may be taken together with X toform a heterocyclic ring;

Z will usually be aromatic, including heterocyclic aromatic, of fromabout 4 to 12, usually 4 to 10 carbon atoms and 0 to 4 heteroatoms, asdescribed above, being bonded directly to C_(β) or through a heteroatom,as described above;

n is 1 or 2, depending upon whether the e-tag moiety is bonded to C_(α)or X;

wherein one of Y and Z will have a functionality for binding to thebinding moiety, or be bound to the binding moiety, e.g. by serving as,or including a linkage group, to a binding moiety, T.

Preferably, W, X, Y, and Z are selected so that upon cleavageelectrophoretic tag, E, is within the size limits described below.

While not depicted in the formula, one may have a plurality of e-tagmoieties in a single molecule, by having one or more a-tag moietiesjoined to one or both Xs.

Illustrative cleavable linkages include S-3-thiolacrylic acid,—N,N-methyl 4-amino-4-butenoic acid, —O, 3-hydroxyacrolein,N-(4-carboxyphenyl) 2-imidazole, oxazole, and thiazole.

Also of interest are N-alkyl acridinyl derivatives, substituted at the 9position with a divalent group of the formula:—(CO)X¹(A)-wherein:

X¹ is a heteroatom selected from the group consisting of O, S, N, andSe, usually one of D the first three; and

A is a chain of at least 2 carbon atoms and usually not more than 6carbon atoms substituted with an e-tag reporter, where preferably theother valences of A are satisfied by hydrogen, although the chain may besubstituted with other groups, such as alkyl, aryl, heterocyclic groups,etc., A generally being not more than 10 carbon atoms.

Also of interest are heterocyclic compounds, such asdiheterocyclopentadienes, as exemplified by substituted imidazoles,thiazoles, oxazoles, etc., where the rings will usually be substitutedwith at least one aromatic group and in some instances hydrolysis willbe necessary to release the e-tag reporter.

Also of interest are tellurium (Te) derivatives, where the Te is bondedto an ethylene group having a hydrogen atom β to the Te atom, whereinthe ethylene group is part of an alicyclic or heterocyclic ring, thatmay have an oxo group, preferably fused to an aromatic ring and theother valence of the Te is bonded to the e-tag reporter. The rings maybe coumarin, benzoxazine, tetralin, etc.

Several preferred cleavable linkages and their cleavage products areillustrated in FIGS. 10 A-F. The thiazole cleavable linkage,“—CH₂-thiazole-(CH2)_(n)—C(═O)—NH-protein,” shown in FIG. 10A, resultsin an electrophoretic tag with the moiety “—CH₂—C(═O)—NH—CHO.”Preferably, n is in the range of from 1 to 12, and more preferably, from1 to 6. The oxazole cleavable linkage,“—CH₂-oxazole-(CH2)_(n)C(═O)—NH-protein,” shown in FIG. 10B, results inan electrophoretic tag with the moiety “—CH₂—C(═O)O—CHO.” An olefincleavable linkage (FIG. 10C) is shown in connection with theelectrophoretic probe embodiment “T-L-M-D,” described above and with Dbeing a fluorescein dye. The olefin cleavable linkage may be employed inother embodiments also. Cleavage of the illustrated olefin linkageresults in an electrophoretic tag of the form: “R—(C═O)-M-D,” where “R”may be any substituent within the general description of theelectrophoretic tags, E, provided above. Preferably, R is anelectron-donating group, e.g. Ullman et al, U.S. Pat. No. 6,251,581;Smith and March, March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure, 5^(th) Edition (Wiley-Interscience, New York,2001); and the like. More preferably, R is an electron-donating grouphaving from 1-8 carbon atoms and from 0 to 4 heteroatoms selected fromthe group consisting of O, S, and N. In further preference, R is —N(O)₂,—OQ, p-[C₆H₄N(Q)₂], furanyl, n-alkylpyrrolyl, 2-indolyl, or the like,where D is alkyl or aryl. In further reference to the olefin cleavablelinkage of FIG. 10C, substituents “X” and “R” are equivalent tosubstituents “X” and “Y” of the above formula describing cleavablelinkage, L. In particular, X in FIG. 10C is preferably morpholino, —OR′,or —SR″, where R′ and R″ are aliphatic, aromatic, alicyclic orheterocyclic having from 1 to 8 carbon atoms and 0 to 4 heteroatomsselected from the group consisting of O, S, and N. A preferred thioethercleavable linkage is illustrated in FIG. 10D having the form“—(CH₂)₂—S—CH(C₆H₅)C(═O)NH—(CH₂)_(n)NH—,” wherein n is in the range offrom 2 to 12, and more preferably, in the range of from 2 to 6.Thioether cleavable linkages of the type shown in FIG. 10D may beattached to binding moieties, T, and electrophoretic tags, E, by way ofprecursor compounds shown in FIGS. 10E and 10F. To attach to an aminogroup of a binding moiety, T, the terminal hydroxyl is converted to anNHS ester by conventional chemistry. After reaction with the amino groupand attachment, the Fmoc protection group is removed to produce a freeamine which is then reacted with an NHS ester of the e-tag, such ascompounds produced by the schemes of FIGS. 1, 2, and 4, with theexception that the last reaction step is the addition of an NHS ester,instead of a phosphoramidite group.

Electrophoretic tag, E, is a water soluble organic compound that isstable with respect to the active species, especially singlet oxygen,and that includes a detection or reporter group. Otherwise, E may varywidely in size and structure. Preferably, E carries a charge at neutralpH and has a molecular weight in the range of from about 150 to about10,000 daltons, more preferably, from about 150 to about 5000 daltons,and most preferably, from about 150 to 2500 daltons. Preferredstructures of E are described more fully below. Preferably, thedetection group generates an electrochemical, fluorescent, orchromogenic signal. Most preferably, the detection group generates afluorescent signal. Compositions of the invention include pluralities ofelectrophoretic tags that may be used together to carry out themultiplexed assays of the invention. Preferably, the plurality ofelectrophoretic tags in a composition is at least 5, and morepreferably, at least 10. Still more preferably, the plurality is in therange of from 5 to 200, and more preferably, from 5 to 100, or 5 to 75,or from 5 to 50, or from 10 to 30. Preferably, electrophoretic tagswithin a plurality of a composition each have either a uniquecharge-to-mass ratio and/or a unique optical property with respect tothe other members of the same plurality. Preferably, the opticalproperty is a fluorescence property, such as emission spectrum,fluorescence lifetime, or the like. More preferably, the fluorescenceproperty is emission spectrum. For example, each electrophoretic tag ofa plurality may have the same fluorescent emission properties, but eachwill differ from one another by virtue of unique charge-to-mass ratios.On the other hand, or two or more of the electrophoretic tags of aplurality may have identical charge-to-mass ratios, but they will haveunique fluorescent properties, e.g. spectrally resolvable emissionspectra, so that all the members of the plurality are distinguishable bythe combination of electrophoretic separation and fluorescencemeasurement.

Preferably, electrophoretic tags in a plurality are detected byelectrophoretic separation and fluorescence. Preferably, electrophoretictags having substantially identical fluorescence properties havedifferent electrophoretic mobilities so that distinct peaks in anelectropherogram are formed under separation conditions. A measure ofthe distinctness, or lack of overlap, of adjacent peaks iselectrophoretic resolution, which is the distance between adjacent peakmaximums divided by four times the larger of the two standard deviationsof the peaks. Preferably, adjacent peaks have a resolution of at least1.0, and more preferably, at least 1.5, and most preferably, at least2.0. In a given separation and detection system, the desired resolutionmay be obtained by selecting a plurality of electrophoretic tags whosemembers have electrophoretic mobilities that differ by at least apeak-resolving amount, such quantity depending on several factors wellknown to those of ordinary skill, including signal detection system,nature of the fluorescent moieties, the diffusion coefficients of thetags, the presence or absence of sieving matrices, nature of theelectrophoretic apparatus, e.g. presence or absence of channels, lengthof separation channels, and the like. Preferably, pluralities ofelectrophoretic tags of the invention are separated by conventionalcapillary electrophoresis apparatus, either in the presence or absenceof a conventional sieving matix. Exemplary capillary electroresisapparatus include Applied Biosystems (Foster City, Calif.) models 310,3100 and 3700; Beckman (Fullerton, Calif.) model P/ACE MDQ; AmershamBiosciences (Sunnyvale, Calif.) MegaBACE 1000 or 4000; SpectruMedixgenetic analysis system; and the like. Preferably, in such conventionalapparatus, the electrophoretic mobilities of electrophoretic tags of aplurality differ by at least one percent, and more preferably, by atleast a percentage in the range of from 1 to 10 percent.

Electrophoretic mobility is proportional to q/M^(2/3), where q is thecharge on the molecule and M is the mass of the molecule. Desirably, thedifference in mobility under the conditions of the determination betweenthe closest electrophoretic labels will be at least about 0.001, usually0.002, more usually at least about 0.01, and may be 0.02 or more.

A preferred structure of electrophoretic tag, E, is (M, D), where M is amobility-modifying moiety and D is a detection moiety. The notation “(M,D)” is used to indicate that the ordering of the M and D moieties may besuch that either moiety can be adjacent to the cleavable linkage, L.That is, “T-L-(M, D)” designates electrophoretic probe of either of twoforms: “T-L-M-D” or “T-L-D-M.”

Detection moiety, D, may be a fluorescent label or dye, a chromogeniclabel or dye, an electrochemical label, or the like. Preferably, D is afluorescent dye. Exemplary fluorescent dyes for use with the inventioninclude water-soluble rhodamine dyes, fluorescein,4,7-dichlorofluoresceins, benzoxanthene dyes, and energy transfer dyes,disclosed in the following references: Handbook of Molecular Probes andResearch Reagents, 8th ed., (Molecular Probes, Eugene, 2002); Lee et al,U.S. Pat. No. 6,191,278; Lee et al, U.S. Pat. No. 6,372,907; Menchen etal, U.S. Pat. No. 6,096,723; Lee et al, U.S. Pat. No. 5,945,526; Lee etal, Nucleic Acids Research, 25: 2816-2822 (1997); Hobb, Jr., U.S. Pat.No. 4,997,928; Khanna et al., U.S. Pat. No. 4,318,846; Reynolds, U.S.Pat. No. 3,932,415; Eckert et al, U.S. Pat. No. 2,153,059; Eckert et al,U.S. Pat. No. 2,242,572; Thing et al, International patent publicationWO 02/30944; and the like. Further specific exemplary fluorescent dyesinclude 5- and 6-carboxyrhodamine 6G; 5- and 6-carboxy-X-rhodamine, 5-and 6-carboxytetramethylrhodamine, 5- and 6-carboxyfluorescein, 5- and6-carboxy 4,7-dichlorofluorescein, 2′,T-dimethoxy-5- and6-carboxy-4,7-dichlorofluorescein, 2′,7′-dimethoxy-4′,5′-dichloro-5- and6-carboxyfluorescein, 2′,7′-dimethoxy-4′,5′-dichloro-5- and6-carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-5- and6-carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-4′,5′-dichloro-5-and 6-carboxy-4,7-dichlorofluorescein, 2′,7′-dichloro-5- and6-carboxy-4,7-dichlorofluorescein, and 2′,4′,5′,7′-tetrachloro-5- and6-carboxy-4,7 dichlorofluorescein. Most preferably, D is a fluoresceinor a fluorescein derivative. Exemplary most preferred dyes for use withthe invention are shown in FIGS. 6A and 6B.

M is generally a chemical group or moiety that has or is designed tohave a particular charge-to-mass ratio, and thus a particularelectrophoretic mobility in a defined electrophoretic system. Exemplarytypes of mobility-modifying moieties are discussed below. In a set of nelectrophoretic probes, each unique mobility modifier is designatedM_(j), where j=1 to n, and n has a value as described above. Themobility-modifying moiety may be considered to include a mass-modifyingregion and/or a charge-modifying region or a single region that acts asboth a mass- and charge-modifying region. In the probe sets utilized inthe invention, the mobility-modifying moiety may have one or more of thefollowing characteristics: (i) a unique charge-to-mass ratio due tovariations in mass, but not charge; (ii) a unique charge-to-mass ratiodue to changes in both mass and charge; and (iii) a uniquecharge-to-mass ratios of between about −0.0001 and about 0.5, usually,about −0.001 and about 0.1. As noted above, D is typically common amonga set or plurality of different electrophoretic probes, but may alsodiffer among probe sets, contributing to the unique electrophoreticmobilities of the released a-tag.

The size and composition of mobility-modifying moiety, M, can vary froma bond to about 100 atoms in a chain, usually not more than about 60atoms, more usually not more than about 30 atoms, where the atoms arecarbon, oxygen, nitrogen, phosphorous, boron and sulfur. Generally, whenother than a bond, the mobility-modifying moiety has from about 0 toabout 40, more usually from about 0 to about 30 heteroatoms, which inaddition to the heteroatoms indicated above may include halogen or otherheteroatom. The total number of atoms other than hydrogen is generallyfewer than about 200 atoms, usually fewer than about 100 atoms. Whereacid groups are present, depending upon the pH of the medium in whichthe mobility-modifying moiety is present, various cations may beassociated with the acid group. The acids may be organic or inorganic,including carboxyl, thionocarboxyl, thiocarboxyl, hydroxamic, phosphate,phosphite, phosphonate, phosphinate, sulfonate, sulfinate, boronic,nitric, nitrous, etc. For positive charges, substituents include amino(includes ammonium), phosphonium, sulfonium, oxonium, etc., wheresubstituents are generally aliphatic of from about 1-6 carbon atoms, thetotal number of carbon atoms per heteroatom, usually be less than about12, usually less than about 9. The side chains include amines, ammoniumsalts, hydroxyl groups, including phenolic groups, carboxyl groups,esters, amides, phosphates, heterocycles. M may be a homo-oligomer or ahetero-oligomer, having different monomers of the same or differentchemical characteristics, e.g., nucleotides and amino acids.

The charged mobility-modifying moieties generally have only negative orpositive charges, although one may have a combination of charges,particularly where a region to which the mobility-modifying moiety isattached is charged and the mobility-modifying moiety has the oppositecharge. The mobility-modifying moieties may have a single monomer thatprovides the different functionalities for oligomerization and carry acharge or two monomers may be employed, generally two monomers. One mayuse substituted diols, where the substituents are charged and dibasicacids. Illustrative of such oligomers is the combination of diols ordiamino, such as 2,3-dihydroxypropionic acid, 2,3-dihydroxysuccinicacid, 2,3-diaminosuccinic acid, 2,4-dihydroxyglutaric acid, etc. Thediols or diamino compounds can be linked by dibasic acids, which dibasicacids include the inorganic dibasic acids indicated above, as well asdibasic acids, such as oxalic acid, malonic acid, succinic acid, maleicacid, furmaric acid, carbonic acid, etc. Instead of using esters, onemay use amides, where amino acids or diamines and diacids may beemployed. Alternatively, one may link the hydroxyls or amines withalkylene or arylene groups.

By employing monomers that have substituents that provide for charges,or which may be modified to provide charges, one can provide formobility-modifying moieties having the desired charge-to-mass ratio. Forexample, by using serine or threonine, one may modify the hydroxylgroups with phosphate to provide negatively charged mobility-modifyingmoieties. With arginine, lysine and histidine, one provides forpositively charged mobility-modifying moieties. Oligomerization may beperformed in conventional ways to provide the appropriately sizedmobility-modifying moiety. The different mobility-modifying moietieshaving different orders of oligomers, generally having from 1 to 20monomeric units, more usually about 1 to 12, where a unit intends arepetitive unit that may have from 1 to 2 different monomers. For themost part, oligomers may be used with other than nucleic acidtarget-binding regions. The polyfunctionality of the monomeric unitsprovides for functionalities at the termini that may be used forconjugation to other moieties, so that one may use the availablefunctionality for reaction to provide a different functionality. Forexample, one may react a carboxyl group with an aminoethylthiol, toreplace the carboxyl group with a thiol functionality for reaction withan activated olefin.

By using monomers that have about 1 to about 3 charges, one may employ alow number of monomers and provide for mobility variation with changesin molecular weight. Of particular interest are polyolpolycarboxylicacids having from about two to four of each functionality, such astartaric acid, 2,3-dihydroxyterephthalic acid, 3,4-dihydroxyphthalicacid, A5-tetrahydro-3,4-dihydroxyphthalic acid, etc. To provide for anadditional negative charge, these monomers may be oligomerized with adibasic acid, such as a phosphoric acid derivative to form the phosphatediester. Alternatively, the carboxylic acids could be used with adiamine to form a polyamide, while the hydroxyl groups could be used toform esters, such as phosphate esters, or ethers such as the ether ofglycolic acid, etc. To vary the mobility, various aliphatic groups ofdiffering molecular weight may be employed, such as polymethylenes,polyoxyalkylenes, polyhaloaliphatic or aromatic groups, polyols, e.g.,sugars, where the mobility will differ by at least about 0.01, moreusually at least about 0.02 and more usually at least about 0.5.

In another aspect, (M,D) moieties are constructed from chemicalscaffolds used in the generation of combinatorial libraries. Forexample, the following references describe scaffold compound useful ingenerating diverse mobility modifying moieties: peptoids (PCTPublication No WO 91/19735, Dec. 26, 1991), encoded peptides (PCTPublication WO 93/20242, Oct. 14 1993), random bio-oligomers (PCTPublication WO 92/00091, Jan. 9, 1992), benzodiazepines (U.S. Pat. No.5,288,514), diversomeres such as hydantoins, benzodiazepines anddipeptides (Hobbs DeWitt, S. et al., Proc. Nat. Acad. Sci. U.S.A. 90:6909-6913 (1993), vinylogous polypeptides (Hagihara et al. J. Amer.Chem. Soc. 114: 6568 (1992)), nonpeptidal peptidomimetics with aBeta-D-Glucose scaffolding (Hirschmann, R. et al., J. Amer. Chem. Soc.114: 9217-9218 (1992)), analogous organic syntheses of small compoundlibraries (Chen, C. et al. J. Amer. Chem. Soc. 116: 2661 (1994)),oligocarbamates (Cho, C. Y. et al. Science 261: 1303 (1993)), peptidylphosphonates (Campbell, D. A. et al., J. Org. Chem. 59:658 (1994));Cheng et al, U.S. Pat. No. 6,245,937; Heizmann et al, “Xanthines as ascaffold for molecular diversity,” Mol. Divers. 2: 171-174 (1997); Paviaet al, Bioorg. Med. Chem., 4: 659-666 (1996); Ostresh et al, U.S. Pat.No. 5,856,107; Gordon, E. M. et al., J. Med. Chem. 37: 1385 (1994); andthe like. Preferably, in this aspect, D is a substituent on a scaffoldand M is the rest of the scaffold.

In yet another aspect, (M, D) moieties are constructed from one or moreof the same or different common or commercially available linking,cross-linking, and labeling reagents that permit facile assembly,especially using a commercial DNA or peptide synthesizer for all or partof the synthesis. In this aspect, (M, D) moieties are made up ofsubunits usually connected by phosphodiester and amide bonds. Exemplary,precusors include, but are not limited to, dimethoxytrityl(DMT)-protected hexaethylene glycol phosphoramidite, 6-(4Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,12-(4-Monomethoxytritylamino)dodecyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl),N,N-diisopropyl)-phosphoramidite, (STrityl-6-mercaptohexyl)-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,5′-Fluorescein phosphoramidite, 5′-Hexachloro-FluoresceinPhosphoramidite, 5′-Tetrachloro-Fluorescein Phosphoramidite,9-O-Dimethoxytrityl-triethylene glycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,3(4,4-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,5′-O-Dimethoxytrityl-1′,2′-Dideoxyribose-3′-[(2-cyanoethyl)-(N,Ndiisopropyl)]-phosphoramidite, 18-0 Dimethoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,12-(4,4′-Dimethoxytrityloxy)dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,1,3-bis-[5-(4,4′-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,1-[5-(4,4′-dimethoxytrityloxy)pentylamido]-3-[5-fluorenomethoxycarbonyloxypentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,Tris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC),succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidylacetylthioacetate, Texas Red-X-succinimidyl ester, 5- and6-carboxytetramethylrhodamine succinimidyl ester, bis-(4carboxypiperidinyl)sulfonerhodamine di(succinimidyl ester), 5- and6-((N-(5-aminopentyl)aminocarbonyl)tetramethylrhodamine, succinimidyl4-(p-maleimidophenyl)butyrate (SMPB);N-y-maleimidobutyryl-oxysuccinimide ester (GMBS); p-nitrophenyliodoacetate (NPIA); 4-(4-N-maleimidophenyl)butyric acid hydrazide(MPBH); and like reagents. The above reagents are commerciallyavailable, e.g. from Glen Research (Sterling, Va.), Molecular Probes(Eugene, Oreg.), Pierce Chemical, and like reagent providers. Use of theabove reagents in conventional synthetic schemes is well known in theart, e.g. Hermanson, Bioconjugate Techniques (Academic Press, New York,1996). In particular, M may be constructed from the following reagents:dimethoxytrityl (DMT)-protected hexaethylene glycol phosphoramidite,6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,1244Monomethoxytritylamino)dodecyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl),N,N-diisopropyl)-phosphoramidite,(S-Trityl-6-mercaptohexyl)-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,9-0-Dimethoxytrityl-triethylene glycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,3(4,4′Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,5′-O-Dimethoxytrityl-1′,2′-Dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,18-0 Dimethoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,12-(4,4′-Dimethoxytrityloxy)dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,1,3-bis-[5-(4,4′-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,1-[5-(4,4′-dimethoxytrityloxy)pentylamido]-3-[5-fluorenomethoxycarbonyloxypentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,Tris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC),succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidylacetylthioacetate, succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB);N-y-maleimidobutyryl-oxysuccinimide ester (GMBS); p-nitrophenyliodoacetate (NPIA); and 4-(4-N-maleimidophenyl)butyric acid hydrazide(MPBH).

M may also comprise polymer chains prepared by known polymer subunitsynthesis methods. Methods of forming selected-length polyethyleneoxide-containing chains are well known, e.g. Grossman et al, U.S. Pat.No. 5,777,096. It can be appreciated that these methods, which involvecoupling of defined-size, multi-subunit polymer units to one another,directly or via linking groups, are applicable to a wide variety ofpolymers, such as polyethers (e.g., polyethylene oxide and polypropyleneoxide), polyesters (e.g., polyglycolic acid, polylactic acid),polypeptides, oligosaccharides, polyurethanes, polyamides,polysulfonamides, polysulfoxides, polyphosphonates, and block copolymersthereof, including polymers composed of units of multiple subunitslinked by charged or uncharged linking groups. In addition tohomopolymers, the polymer chains used in accordance with the inventioninclude selected-length copolymers, e.g., copolymers of polyethyleneoxide units alternating with polypropylene units. As another example,polypeptides of selected lengths and amino acid composition (i.e.,containing naturally occurring or man-made amino acid residues), ashomopolymers or mixed polymers.

In another aspect, the detection moiety of (M,D) generates a fluorescentsignal by an energy transfer mechanism. Preferably, in this aspect, Dhas the form “D₁-g-D₂” where D₁ and D₂ are acceptor-donor pairs ofmolecules, e.g. Wu et al, Anal. Biochem., 218: 1-13 (1994), and g is arigid linker that maintains D₁ and D₂ at a substantially constantdistance. Guidance in selecting rigid linker, g, may be found in We etal (cited above) and in U.S. Pat. Nos. 5,863,727; 5,800,996; 5,945,526;and 6,008,379. Either D₁ or D₂ may be the acceptor and the other thedonor molecule in the pair. Exemplary, energy transfer detectionmoieties for use with the invention are disclosed in Lee et al, U.S.Pat. No. 5,945,526; Lee et al, Nucleic Acids Research, 25: 2816-2822(1997); Taing et al, International patent publication WO 02/30944; andlike references. Preferably, rigid linker, g, is selected so that thedistance between D₁, and D₂ is maintained at a substantially constantdistance within the range of from 10-100 Angstroms. A wide variety oflinking groups may be employed with the proviso that the linkage bestable to the presence of singlet oxygen. Preferably, D₁, and D₂ areselected from the set of fluorescein, rhodamine, rhodamine 6G, rhodamine110, rhodamine X, tetramethylrhodamine, and halogenated derivativesthereof. More preferably, D₁, and D₂ are both fluorescein dyes.

In one aspect, g may be selected from any of R₁—R₂—R₁ andR₁—R₂—C(═O)—X₁—R₃, the latter being present in either orientation withrespect to D₁, and D₂; where X₁ is O, S, or NH; R₁ is (C₁-C₅ alkyldiyl,X₁, C(═O)) such that any one to three the moieties in parentheses arearranged in any linear order; R₂ is a 5 to 6 membered ring selected fromthe group consisting of cyclopentene, cyclohexene, cyclopentadiene,cyclohexadiene, furan, pyrrole, isopyrole, isoazole, pyrazole,isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine,pyrazine oxazine, indene, benzofuran, thionaphthene, indole andnaphthalene; R₃ is C₁-C₅ alkyldiyl.

In a preferred aspect, after release, electrophoretic tag, E, is definedby the formula:A-M-Dwherein:

A is —C(═O)R, where R is aliphatic, aromatic, alicyclic or heterocyclichaving from 1 to 8 carbon atoms and 0 to 4 heteroatoms selected from thegroup consisting of O, S, and N; —CH₂—C(═O)—NH—CHO; —SO₂H;—CH₂—C(═O)O—CHO; —C(═O)NH—(CH₂)_(n)—NH—C(═O)C(═O)—(C₆H₅), where n is inthe range of from 2 to 12;

D is a fluorescent dye; and

M is as described above, with the proviso that the total molecularweight of A-M-D be within the range of from about 150 to about 5000daltons.

In a preferred aspect, D is a fluorescein, e.g. as described above andillustrated in FIGS. 6A and 6B, and the total molecular weight of A-M-Dis in the range of from about 150 to about 2500 daltons.

In another preferred aspect, D is of the form “D₁-g-D₂” as describedabove.

In some embodiments the e-tag moieties need not be charged but merelydiffer in mass. Thus, one could use the same or similar monomers, wherethe functionalities would be neutral or made neutral, such as esters andamides of carboxylic acids. Also, one may vary the e-tag moieties byisotopic substitution, such as ²H, ¹⁸O, ¹⁴c, etc.

Pluralities of electrophoretic tags may include oligopeptides forproviding the charge, particularly oligopeptides of from 2-6, usually2-4 monomers, either positive charges resulting from lysine, arginineand histidine or negative charges, resulting from aspartic and glutamicacid. Of course, one need not use naturally occurring amino acids, butunnatural or synthetic amino acids, such as taurine, phosphatesubstituted serine or threonine, S-a-suceinylcysteine, co-oligomers ofdiamines and amino acids, etc.

In one embodiment of the present invention, the charge-imparting moietyis conveniently composed primarily of amino acids but also may includethioacids and other carboxylic acids having from one to five carbonatoms. The charge imparting moiety may have from about 1 to about 30,preferably about 1 to about 20, more preferably, about 1 to about 10amino acids per moiety and may also comprise about 1 to about 3thioacids or other carboxylic acids. However, when used with anuncharged sub-region, the charged sub-region will generally have fromabout 1 to about 4, frequently about 1 to about 3 amino acids. Asmentioned above, any amino acid, both naturally occurring and synthetic,may be employed.

In a particular embodiment, T-L-M-D may be represented by the formula:T-L-(amino acid)_(n)-L′-Fluorescerwherein L′ is a bond or a linking group of from 1 to 20 atoms other thanhydrogen, n is I to 20, and L is a cleavable linkage to thepolypeptide-binding moiety. In this embodiment T is linked to theterminal amino acid by a cleavable linkage. An example of thisembodiment, by way of illustration and not limitation, is one in whichthe fluorescer is fluorescein, L′ is a bond in the form of an amidelinkage involving the meta-carboxyl of the fluorescein and the terminalamine group of lysine, and T is a polypeptide-binding moiety.

Examples of electrophoretic tags based on such label conjugates may berepresented as follows:Fluorescein-(CO)NH—(CH₂)₄—NH-(amino acid)_(n)where formulas and charges at neutral pH for specific compounds are setforth in Table 2.

TABLE 2 No. (amino acid)n Charge (q) Mol. Wt. (M) q/M^(2/3)  1 none −1 446 −.0178  2 lysine −1  591 −.0148  3 (lysine)₂ neutral  737   .0128 4 alanine −2  534 −.0298  5 aspartic acid −3  578 −.0423  6 (asparticacid)₂ −4  711 −.0491  7 (aspartic acid)₃ −5  844 −.0877  8 (asparticacid)₄ −6  977 −.0595  9 (aspartic acid)₅ −7 1110 −.0638 10 (asparticacid)₆ −8 1243 −.0675 11 (aspartic acid)₇ −9 1376 −.0710 12alanine-lysine −2  680 −.0253 13 aspartic acid-lysine −2  724 −.0243 14(aspartic acid)₂-lysine −3  857 −.0325 15 (aspartic acid)₃-lysine −4 990 −.0393 16 (aspartic acid)₄-lysine −5 1123 −.0452 17 (asparticacid)₅-lysine −6 1256 −.0503 18 (aspartic acid)₆-lysine −7 1389 −.054919 (aspartic acid)₇-lysine −8 1522 −.0590 20 (aspartic acid)₈-lysine −91655 −.0627 21 (lysine)₄ +2 1029   .0192 22 (lysine)₅ +3 1170   .0264

wherein q is charge, M is mass and mobility is proportional to q/MV3.

In another embodiment, mobility-modifying moiety, M, is dependent onusing an alkylene or aralkylene (comprising a divalent aliphatic grouphaving about 1 to about 2 aliphatic regions and about 1 to about 2aromatic regions, generally benzene), where the groups may besubstituted or unsubstituted, usually unsubstituted, of from about 2 toabout 16, more usually about 2 to about 12, carbon atoms, where themobility-modifying moiety may link the same or different fluorescers toa monomeric unit, e.g., a nucleotide. The mobility-modifying moiety mayterminate in a carboxy, hydroxy or amino group, being present as anester or amide. By varying the substituents on the fluorophore, one canvary the mass in units of at least about 5 or more, usually at leastabout 9, so as to be able to obtain satisfactory separation in capillaryelectrophoresis. To provide further variation, a thiosuccinimide groupmay be employed to join alkylene or aralkylene groups at the nitrogenand sulfur, so that the total number of carbon atoms may be in the rangeof about 2 to about 30, more usually about 2 to about 20. Instead of orin combination with the above groups and to add hydrophilicity, one mayuse alkyleneoxy groups.

Besides the nature of the mobility-modifying moiety, as alreadyindicated, diversity can be achieved by the chemical and opticalcharacteristics of the label, the use of energy transfer complexes,variation in the chemical nature of the mobility-modifying moiety, whichaffects mobility, such as folding, interaction with the solvent and ionsin the solvent, and the like. In one embodiment of the invention, themobility-modifying moiety may be an oligomer, where themobility-modifying moiety may be synthesized on a support or produced bycloning or expression in an appropriate host. Conveniently, polypeptidescan be produced where there is only one cysteine orserine/threonine/tyrosine, aspartic/glutamic acid, orlysine/arginine/histidine, other than an end group, so that there is aunique functionality, which may be differentially functionalized. Byusing protective groups, one can distinguish a side-chain functionalityfrom a terminal amino acid functionality. Also, by appropriate design,one may provide for preferential reaction between the samefunctionalities present at different sites on the mobility-modifyingmoiety. Whether one uses synthesis or cloning for preparation ofoligopeptides, is to a substantial degree depend on the length of themobility-modifying moiety.

Substituted aryl groups can serve as both mass- and charge-modifyingregions. Various functionalities may be substituted onto the aromaticgroup, e.g., phenyl, to provide mass as well as charges to the e-tagreporter. The aryl group may be a terminal group, where only one linkingfunctionality is required, so that a free hydroxyl group may beacylated, may be attached as a side chain to an hydroxyl present on thee-tag reporter chain, or may have two functionalities, e.g., phenolichydroxyls, that may serve for phosphite ester formation and othersubstituents, such as halo, haloalkyl, nitro, cyan, alkoxycarbonyl,alkylthio, etc. where the groups may be charged or uncharged.

The label conjugates may be prepared utilizing conjugating techniquesthat are well known in the art. M may be synthesized from smallermolecules that have functional groups that provide for linking of themolecules to one another, usually in a linear chain. Such functionalgroups include carboxylic acids, amines, and hydroxy- or thiol-groups.In accordance with the present invention the charge-imparting moiety mayhave one or more side groups pending from the core chain. The sidegroups have a functionality to provide for linking to a label or toanother molecule of the charge-imparting moiety. Common functionalitiesresulting from the reaction of the functional groups employed areexemplified by forming a covalent bond between the molecules to beconjugated. Such functionalities are disulfide, amide, thioamide,dithiol, ether, urea, thiourea, guanidine, azo, thioether, carboxylateand esters and amides containing sulfur and phosphorus such as, e.g.,sulfonate, phosphate esters, sulfonamides, thioesters, etc., and thelike.

The linkages of the components of the e-tag moiety are discussed above.The linkage between the detectable moiety and the mobility-modifyingmoiety is generally stable to the action of the cleavage-inducingmoiety, so that the mobility-modifying moiety and detectable moiety maybe released as an intact unit from the e-tag probe during the cleavageof the e-tag reporter from the e-tag probe.

For the most part, the mobility-modifying moiety may be a bond, wherethe detectable moiety or label is directly bonded to the target-bindingmoiety, or a link of from about 1 to about 35 500 or more, usually about1 to about 300 atoms, more usually about 2 to about 100 atoms in thechain. In this embodiment, the total number of atoms in the chain willdepend to a substantial degree on the diversity required to recognizeall the targets to be determined. The chain of the mobility-modifyingmoiety for the most part is comprised of carbon, nitrogen, oxygen,phosphorous, boron, and sulfur. Various substituents may be present onthe mobility-modifying moiety, which may be naturally present as part ofthe naturally occurring monomer or introduced by synthesis.Functionalities which may be present in the chain include amides,phosphate esters, ethers, esters, thioethers, disulfides, borate esters,sulfate esters, etc. The side chains include amines, ammonium salts,hydroxyl groups, including phenolic groups, carboxyl groups, esters,amides, phosphates, heterocycles, particularly nitrogen heterocycles,such as the nucleoside bases and the amino acid side chains, such asimidazole and quinoline, thioethers, thiols, or other groups of interestto change the mobility of the electrophoretic tag.

The mobility-modifying moiety may be a homooligomer or a heterooligomer,having different monomers of the same or different chemicalcharacteristics, e.g., nucleotides and amino acids. In one embodiment,the e-tag moieties will have a linker, which provides the linkagebetween the mobility-modifying moiety and the detectable label molecule,usually a fluorescer, or a functionality that may be used for linking toa detectable label molecule. By having different functionalities, whichmay be individually bonded to a detectable label molecule, one enhancesthe opportunity for diversity of the electrophoretic tags. Usingdifferent fluorescers for joining to the different functionalities, thedifferent fluorescers can provide differences in light emission andcharge-to-mass ratios for the electrophoretic tags.

Attaching Multiple Electrophoretic Tags to Binding Moieties

In assays involving polypeptides, it is advantageous to have the releaseof multiple e-tag reporters for a binding event involving an individualtarget molecule. In a sense, this results in an amplification of signal.Desirably, the number of e-tag reporters released for each such bindingevent is about 6×10³ to about 6×10¹⁰, preferably, about 6×10⁴ to about6×10⁸. Where the polypeptide-binding moiety has a plurality of sites forattachment such as, for example, an antibody, there is a plurality ofbinding sites on the antibody for attachment of e-tag moieties. When thepolypeptide-binding moiety of the e-tag probe binds to the polypeptideand the first binding moiety of the first binding agent binds to theinduced binding site on the polypeptide thus bringing acleavage-inducing moiety into close proximity to the cleavable linkage,a plurality of e-tag reporters is released for a binding event involvinga single polypeptide. For example, attachment of e-tag moieties to anantibody may result in about 2 to about 10 molecules of e-tag moietiesper antibody molecule.

To further enhance the number of e-tag reporters released, the e-tagmoieties are cleavably attached to a hub, to which a polypeptide-bindingmoiety of the e-tag probe is also attached in a relatively permanentmanner. For a polypeptide-binding moiety that has a plurality ofattachment sites, a plurality of hubs may be attached to thepolypeptide-binding moiety where each hub has a plurality of e-tagmoieties for release. The hub nucleus is, therefore, a polyfunctionalmaterial, normally polymeric, having a plurality of functional groups,e.g., hydroxy, amino, mercapto, carboxy, ethylenic, aldehyde, etc., assites for linking. The functionalities on the hub should be those thatare reactive with a functionality on the e-tag moiety or thepolypeptide-binding moiety to be attached. Some functionalities arepreferred over others because of their ability to resist participationin unwanted side reactions. The hub nucleus may be water soluble orwater insoluble. The hub nucleus is usually at least about 35,000molecular weight and may be about 10 million or more molecular weight,but usually under about 600,000, more usually under about 300,000.Illustrative hub nuclei include polysaccharides, polypeptides,polynucleotides, ion exchange resins, and the like. The hub is in oneaspect a branched linker, which has multiple sites for attachment ofe-tag moieties. The multiple site linkers have an attachment site forattaching the polypeptide-binding moiety and a plurality of sites forattachment of a plurality of e-tag moieties. Of course, the e-tagmoieties must be attached by means of linkages that comprise afunctionality that is cleavable by the cleavage-inducing moiety inaccordance with the present invention.

In one embodiment the hub nucleus is a hydrophilic polymer, generally,an addition or condensation polymer with multiple functionality topermit the attachment of multiple moieties. One class of polymers thatis useful for the reagents of the present invention comprises thepolysaccharide polymers. Polysaccharides such as dextrans, sepharose,polyribose, polyxylose, and the like may be used. Another class ofpolymers are those that result from the addition polymerization ofsubstituted ethylene or butadiene type monomers, including short chainunsaturated monomers such as propylene, wherein these monomers havesubstituents that are hydrophilic groups or can be derivatized tohydrophilic groups. Suitable hydrophilic groups that may be attached tothe ethylene include hydroxy, carboxy and the ester and amides thereof,amines, and the like. If acrylic acid monomers are used, the acid can bederivatized to suitable reactive groups prior to or subsequent topolymerization. Thus, for example, the ester formed from ethylene glycoland acrylic acid provides a hydroxyl group for derivatization to thecomponents of the e-tag probe. Other suitable polymers include polyallylamines and alcohols such as, for example, polyvinyl alcohol. In additionto utilizing polymers derived from a single monomer, mixed polymers mayalso be employed. In this case, the hydrophilicity may be provided by anon-reactive component such as polyethylene glycol, which is thenfurther polymerized to monomers that bear the appropriate functionalgroups for reaction with the components of the e-tag probe. One suchpolymer is a copolymer of polyethylene glycol with polyvinyl alcohol.One specific example of a hub is dextran to which about 10 to about 300molecules of e-tag moieties may be attached per one molecule of dextran.

A particle may be employed to enhance the number of e-tag moietiespresent in the e-tag probe. The particles may be solid (e.g., comprisedof organic and inorganic polymers or latex), oil droplets (e.g.,hydrocarbon, fluorocarbon, silicon fluid), or vesicles (e.g., syntheticsuch as phospholipid or natural such as cells and organelles). The solidparticles are normally polymers, either addition or condensationpolymers, which are readily dispersible in an assay medium. The e-tagmoieties are linked to the particle by cleavable linkages consistentwith the present invention. In this way about 100 to about 105a-tagmoieties may be linked to a single particle. The particle usually has atleast one polypeptide-binding moiety attached to it. It is also withinthe purview of the present invention to attach multiple dextranmolecules to the particle and to link multiple e-tag moieties to thedextran by means of cleavable linkages as discussed above.

In a particular embodiment of an e-tag probe of the invention, thepolypeptide-binding moiety is an antibody. A number of differentreactions may be used to covalently attach compounds to antibodies. Thishas been accomplished by reaction of the amino acid residues of theantibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the suifhydrylgroups of cysteine and the various moieties of the aromatic amino acids.The conjugation to an antibody may be random or site-directed. Forsite-directed conjugation the linker or mobility-modifying moiety may bejoined in any convenient manner to a unit of the target-binding moiety,such as the Fc portion of an antibody or disulfides in the hinge region.For random conjugation amine groups (e.g., N-terminal or lysine) of theantibody may be employed. Alternatively, carboxylate groups (e.g.,C-terminal, aspartic acid, glutamic acid) may be used. Other examplesinclude thiol groups. A primary consideration in binding to an antibodyis retention of antibody recognition properties or specificity andactivity.

Specific approaches are known for attachment to an antibody. One suchapproach is the carbodiimide reaction to link a carboxy (or amino) groupof a compound to amino (or carboxy) groups of the antibody.Additionally, bifunctional agents such as dialdehydes or imidoestershave been used to link the amino group of a compound to amino groups ofthe antibody molecule. In another approach a Schiff base reaction isemployed to link compounds to antibody molecules. This method involvesthe periodate oxidation of the compound to be linked that containsglycol or hydroxy groups, thus forming an aldehyde that is then reactedwith the antibody molecule. Attachment occurs via formation of a Schiffbase with amino groups of the antibody molecule. Furthermore,isocyanates have been used as coupling agents for covalently attachingcompound to antibodies. Suitable linkers for reaction with oxidizedantibodies or oxidized antibody fragments include those containing anamine selected from the group consisting of primary amine, secondaryamine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine,semicarbazide and thiocarbazide groups. Such reactive functional groupsmay exist as part of the structure of the linker, or may be introducedby suitable chemical modification of linkers not containing such groups.

In a particular approach an antibody containing polysaccharide chains isoxidized to produce reactive aldehyde groups. Such oxidation may beachieved by, for example, periodate as known in the art. Hub moleculessuch as, for example, amino-dextran molecules, having e-tag moietiesattached thereto by cleavable linkages are attached to the aldehydegroups on the antibody by reductive amination forming secondary aminelinkages.

Accordingly, in the present invention one or more hub molecules may beattached to an antibody by one of the aforementioned approaches toachieve relatively permanent linkage under the conditions employed inthe present methods. Of course, consistent with the present inventionthe e-tag moieties are attached to the hub by means of a cleavablelinkage or attached directly to the antibody by means of such cleavablelinkage. Upon the binding of a polypeptide by the first binding agentand by the e-tag probe comprising the antibody, which brings the e-tagprobe in close proximity to the cleavage-inducing reagent, multiplee-tag reporters are released for subsequent detection and for relatingto the presence and/or amount of a polypeptide present.

As used herein, the term “capture ligand,” refers to a group that istypically included within the target-binding moiety portion of an e-tagprobe and is capable of binding specifically to a “capture agent” orreceptor. The interaction between such a capture ligand and thecorresponding capture agent may be used to separate uncleaved e-tagprobes from released e-tag reporters. If desired, the receptor may beused to physically sequester the molecules to which it binds, entirelyremoving intact e-tag probes containing the polypeptide-binding regionor modified polypeptide-binding regions retaining the ligand. Thesemodified polypeptide-binding regions may be as a result of degradationof the starting material, contaminants during the preparation, aberrantcleavage, etc., or other nonspecific degradation products of thepolypeptide binding moiety. As above, a ligand, exemplified by biotin,is attached to the polypeptide-binding region so as to be separated fromthe e-tag reporter upon cleavage.

A receptor for the ligand may be used. Such receptors include natural orsynthetic receptors, such as immunoglobulins, lectins, enzymes, etc.,avidin, and so forth. Desirably, the receptor is positively charged,naturally as in the case of avidin, or is made so, by the addition of apositively charged moiety or moieties, such as ammonium groups, basicamino acids, etc. Avidin binds to the biotin attached to the detectionprobe and its degradation products. Avidin is positively charged, whilethe cleaved electrophoretic tag is negatively charged. Thus theseparation of the cleaved electrophoretic tag from, not only uncleavedprobe, but also its degradation products, is easily achieved by usingconventional separation methods. Alternatively, the receptor may bebound to a solid support or high molecular weight macromolecule, such asa vessel wall, particles, e.g., magnetic particles, cellulose, agarose,etc., and separated by physical separation or centrifugation, dialysis,etc. This method further enhances the specificity of the assay andallows for a higher degree of multiplexing.

As a general matter, one may have two ligands, if the nature of thepolypeptide-binding moiety permits. As described above, one ligand canbe used for sequestering e-tag moieties bound to the polypeptide-bindingregion, retaining the first ligand from products lacking the firstligand. Isolation and concentration of the a-tag moieties bound to amodified polypeptide-binding region lacking the first ligand would thenbe performed. In using the two ligands, one would first combine thereaction mixture with a first receptor for the first ligand for removingpolypeptide-binding region retaining the first ligand. One could eitherseparate the first receptor from the composition or the first receptorwould be retained in the composition, as described. This would befollowed by combining the resulting composition, where thepolypeptide-binding region containing the first ligand is bound to thefirst receptor, with the second receptor, which would serve to isolateor enrich for modified polypeptide-binding region lacking the firstligand, but retaining the second ligand. The second ligand could be thedetectable label; a small molecule for which a receptor is available,e.g., a hapten, or a portion of the a-tag probe could serve as thesecond ligand. After the product is isolated or enriched, the a-tagreporter could be released by denaturation of the receptor, displacementof the product, high salt concentrations and/or organic solvents, etc.

Depending upon the reagent to which the e-tag moiety is attached asdiscussed above, there may be a single a-tag moiety or a plurality ofa-tag moieties, generally ranging from about 1 to about 10⁵, moreusually ranging from about 1 to about 300, more particularly rangingfrom about 1 to about 20 depending on whether or not a hub or particleis employed. The number of e-tag moieties bonded to a singletarget-binding region depends upon the sensitivity required, thesolubility of the a-tag moiety, the effect on the assay of a pluralityof e-tag moieties, and the like.

Synthesis of a-tag Probes

The chemistry for performing the types of syntheses to form thecharge-imparting moiety or mobility modifier as a peptide chain is wellknown in the art. See, for example, Marglin, et al., Ann. Rev. Biochem.(1970) 39:841-866. In general, such syntheses involve blocking, with 35an appropriate protecting group, those functional groups that are not tobe involved in the reaction. The free functional groups are then reactedto form the desired linkages. The peptide can be produced on a resin asin the Merrifield synthesis (Merrifield, J. Am. Chem. Soc. (1980)85:2149-2154 and Houghten et al., Int. J. Pep. Prot. Res. (1980)16:311-320. The peptide is then removed from the resin according toknown techniques.

A summary of the many techniques available for the synthesis of peptidesmay be found in J. M. Stewart, et al., “Solid Phase Peptide Synthesis,W.H. Freeman Co, San Francisco (1969); and J. Meienhofer, “HormonalProteins and Peptides”, (1973), vol. 2, p. 46, Academic Press (NewYork), for solid phase peptide synthesis; and E. Schroder, et al., “ThePeptides”, vol. 1, Academic Press (New York), 1965 for solutionsynthesis.

In general, these methods comprise the sequential addition of one ormore amino acids, or suitably protected amino acids, to a growingpeptide chain. Normally, a suitable protecting group protects either theamino or carboxyl group of the first amino acid. The protected orderivatized amino acid can then be either attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected, under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is then added, andso forth. After all the desired amino acids have been linked in theproper sequence, any remaining protecting groups (and any solid support)are removed sequentially or concurrently, to afford the final peptide.The protecting groups are removed, as desired, according to knownmethods depending on the particular protecting group utilized. Forexample, the protecting group may be removed by reduction with hydrogenand palladium on charcoal, sodium in liquid ammonia, etc.; hydrolysiswith trifluoroacetic acid, hydrofluoric acid, and the like.

For synthesis of e-tag probes employing phosphoramidite, or related,chemistry many guides are available in the literature: Handbook ofMolecular Probes and Research Products, 8^(th) edition (MolecularProbes, Inc., Eugene, Oreg., 2002); Beaucage and Iyer, Tetrahedron, 48:2223-2311 (1992); Molko et al, U.S. Pat. No. 4,980,460; Koster et al,U.S. Pat. No. 4,725,677; Caruthers et al, U.S. Pat. Nos. 4,415,732;4,458,066; and 4,973,679; and the like. Many of these chemistries allowcomponents of the electrophoretic probe to be conveniently synthesizedon an automated DNA synthesizer, e.g. an Applied Biosystems, Inc.(Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer, or the like.

Synthesis of e-tag reagents comprising nucleotides as part of themobility-modifying moiety can be easily and effectively achieved viaassembly on a solid phase support using standard phosphoramiditechemistries. The resulting mobility modifying moiety may be linked tothe label and/or polypeptide-binding moiety as discussed above.

The aforementioned label conjugates with different electrophoreticmobility permit a multiplexed detection of multiple polypeptides havinginduced binding sites. It is, of course, within the purview of thepresent invention to prepare any number of label conjugates forperforming multiplexed determinations.

Exemplary Synthetic Approaches for Electrophoretic Tags

One exemplary synthetic approach is outlined in FIG. 1. Starting withcommercially available 6-carboxy fluorescein, the phenolic hydroxylgroups are protected using an anhydride. Isobutyric anhydride inpyridine was employed but other variants are equally suitable. It isimportant to note the significance of choosing an ester functionality asthe protecting group. This species remains intact throughout thephosphoramidite monomer synthesis as well as during oligonucleotideconstruction. These groups are not removed until the synthesized oligois deprotected using ammonia. After protection the crude material isthen activated in situ via formation of an N-hydroxysuccinimide ester(NHS-ester) using DCC as a coupling agent. The DCU by product isfiltered away and an amino alcohol is added. Many amino alcohols arecommercially available some of which are derived from reduction of aminoacids. When the amino alcohol is of the form “H2N—(CH2)1I—OH,” n is inthe range of from 2 to 12, and more preferably, from 2 to 6. Only theamine is reactive enough to displace N-hydroxysuccinimide. Upon standardextractive workup, a 95% yield of product is obtained. This material isphosphitylated to generate the phosphoramidite monomer. For thesynthesis of additional e-tag moieties, a symmetrical bis-amino alcohollinker is used as the amino alcohol (FIG. 2). As such, the second amineis then coupled with a multitude of carboxylic acid derivatives(exemplified by several possible benzoic acid derivatives shown in FIG.3) prior to the phosphitylation reaction. Using this methodologyhundreds, even thousands of e-tag moieties with varying charge-to-massratios can easily be assembled during probe synthesis on a DNAsynthesizer using standard chemistries.

Alternatively, e-tag moieties are accessed via an alternative strategythat uses 5-aminofluorescein as starting material (FIG. 4). Addition of5-aminofluorescein to a great excess of a diacid dichloride in a largevolume of solvent allows for the predominant formation of themonoacylated product over dimer formation. The phenolic groups are notreactive under these conditions. Aqueous workup converts the terminalacid chloride to a carboxylic acid. This product is analogous to6-carboxyfluorescein, and using the same series of steps is converted toits protected phosphoramidite monomer. There are many commerciallyavailable diacid dichlorides and diacids, which can be converted todiacid dichlorides using SOC12 or acetyl chloride. This methodology ishighly attractive in that a second mobility modifier is used. As such,if one has access to 10 commercial modified phosphoramidites and 10diacid dichlorides and 10 amino alcohols there is a potential for 1000different e-tag moieties. There are many commercial diacid dichloridesand amino alcohols (FIG. 5). These synthetic approaches are ideallysuited for combinatorial chemistry.

The electrophoretic tags constructed with the schemes of FIGS. 1, 2, and4 are further reacted either before or after phosphitylation to attach acleavable linkage, e.g. using chemistry as described below.

The e-tag moiety may be assembled having an appropriate functionality atone end for linking to the polypeptide-binding moieties. A variety offunctionalities can be employed. Thus, the functionalities normallypresent in a peptide, such as carboxy, amino, hydroxy and thiol may bethe targets of a reactive functionality for forming a covalent bond. Thee-tag moieties will be linked in accordance with the chemistry of thelinking group and the availability of functionalities on thepolypeptide-binding moiety. For example, as discussed above forantibodies, and fragments thereof such as Fab′ fragments, specific for apolypeptide, a thiol group 5 will be available for using an activeolefin, e.g., maleimide, for thioether formation. Where lysines areavailable, one may use activated esters capable of reacting in water,such as nitrophenyl esters or pentafluorophenyl esters, or mixedanhydrides as with carbodiimide and half-ester carbonic acid. There isample chemistry for conjugation in the literature, so that for eachspecific situation, there is ample precedent in the literature for theconjugation.

In an illustrative synthesis a diol is employed. Examples of such diolsinclude an alkylene diol, polyalkylene diol, with alkylene of from 2 to3 carbon atoms, alkylene amine or poly(alkylene amine) diol, where thealkylenes are of from 2 to 3 carbon atoms and the nitrogens aresubstituted, for example, with blocking groups or alkyl groups of from1-6 carbon atoms, where one diol is blocked with a conventionalprotecting group, such as a dimethyltrityl group. This group can serveas the mass-modifying region and with the amino groups as the chargemodifying region as well. If desired, the mass modifier can be assembledby using building blocks that are joined through phosphoramiditechemistry. In this way the charge modifier can be interspersed betweenthe mass modifier. For example, a series of polyethylene oxide moleculeshaving 1, 2, 3, n units may be prepared. To introduce a number ofnegative charges, a small polyethylene oxide unit may be employed. Themass and charge-modifying region may be built up by having a pluralityof the polyethylene oxide units joined by phosphate units.Alternatively, by employing a large spacer, fewer phosphate groups wouldbe present, so that without large mass differences, large differences inmass-to-charge ratios may be realized.

The chemistry that is employed is the conventional chemistry used inoligonucleotide synthesis, where building blocks other than nucleotidesare used, but the reaction is the conventional phosphoramidite chemistryand the blocking group is the conventional dimethoxytrityl group. Ofcourse, other chemistries compatible with automated synthesizers canalso be used. However, it is desirable to minimize the complexity of theprocess.

As mentioned above, in one embodiment the hub nucleus is a hydrophilicpolymer, generally, an addition or condensation polymer with multiplefunctionality to permit the attachment of multiple moieties. One classof polymers that is useful for the reagents of the present inventioncomprises the polysaccharide polymers such as dextrans, sepharose,polyribose, polyxylose, and the like. For example, the hub may bedextran to which multiple e-tag reporters may be attached in a cleavablemanner consistent with the present invention. A few of the aldehydemoieties of the dextran remain and may be used to attach the dextranmolecules to amine groups on an oligonucleotide by reductive amination.In another example using dextran as the hub nucleus, the dextran may becapped with succinic anhydride and the resulting material may be linkedto amine-containing oligonucleotides by means of amide formation.

Besides the nature of the linker and mobility-modifying moiety, asalready indicated, diversity can be achieved by the chemical and opticalcharacteristics of the fluorescer, the use of energy transfer complexes,variation in the chemical nature of the linker, which affects mobility,such as folding, interaction with the solvent and ions in the solvent,and the like. As already suggested, in one embodiment the linker is anoligomer, where the linker may be synthesized on a support or producedby cloning or expression in an appropriate host. Conveniently,polypeptides can be produced where there is only one cysteine orserine/threonine/tyrosine, aspartic/glutamic acid, orlysine/arginine/histidine, other than an end group, so that there is aunique functionality, which may be differentially functionalized. Byusing protective groups, one can distinguish a side-chain functionalityfrom a terminal amino acid functionality. Also, by appropriate design,one may provide for preferential reaction between the samefunctionalities present at different sites on the linking group. Whetherone uses synthesis or cloning for preparation of oligopeptides, will toa substantial degree depend on the length of the linker.

For 20 different e-tag reporters, only 5 different mass-modifyingregions, one cleavable link and four different detectable regions arenecessary. For 120 e-tag reporters, one need only have 10 differentmass-modifying regions, 3 different charge-modifying regions and 4different detectable regions. For 500 different e-tag reporters, oneneed only have 25 different mass modifying regions, 5 differentcharge-modifying regions and 4 different detectable regions.

Methods for Use of e-tag Reagents

The following general discussion of methods and examples of specificassays are by way of illustration and not limitation. One skilled in theart will be able to apply the technology herein in assaying for anyanalytes in most assay formats that will be apparent to the skilledartisan particularly protein assays and the area of chemical genetics.

In carrying out the assays, the components, i.e., the sample, the firstreagent and the electrophoretic probes, are combined in an assay mediumin any order, usually simultaneously. Alternatively, one or more of thereagents may be combined with one or more of the remaining agents toform a subcombination. The subcombination can then be subjected toincubation. Then, the remaining reagents or subcombination thereof maybe combined and the mixture incubated. The amounts of the reagents areusually determined empirically. As a general rule, at least an equalamount of the first reagent and the electrophoretic probe is employed tothe highest expected amount of the polypeptides of interest, usually atleast about 1.5 fold excess, more usually at least about 2 fold excessand may have about 10 fold excess or more. The components are combinedunder binding conditions, usually in an aqueous medium, generally at apH in the range of about 5 to about 10, with buffer at a concentrationin the range of about 10 to about 200 mM. These conditions areconventional, where conventional buffers may be used, such as phosphate,carbonate, HEPES, MOPS, Tris, borate, etc., as well as otherconventional additives, such as salts, stabilizers, organic solvents,etc. The aqueous medium may be solely water or may include from 0.01 to80 or more volume percent of a co-solvent.

The combined reagents are incubated for a time and at a temperature thatpermit a substantial number of binding events to occur. Generally, thetime for incubation after combination of all or a portion of thereagents is at least about 5 min, more usually at least about 15 min,before irradiating the mixture or adding the remaining reagents.Moderate temperatures are normally employed for the incubation andusually constant temperature. Incubation temperatures will normallyrange from about 5° to 99° C., usually from about 15° to 70° C., moreusually 20 to 45° C. Temperatures during measurements will generallyrange from about 10° to 70° C., more usually from about 20° to 45° C.,more usually 20° to 25° C.

After the appropriate incubation periods and after all of the reagentshave been combined to form a combination comprising the first reagent orcleavage-inducing reagent, the sample and the electrophoretic probe ore-tag probe, the mixture is treated to activate the cleavage-inducingmoiety. The nature of the activation is, of course, dependent on thenature of the cleavage-30 inducing moiety.

The subject invention employs a variety of reagent systems, where abinding event results in release of an e-tag moiety. The effect of thecleavage-inducing moiety is to make or break a bond by physical,chemical or enzymatic means. Each of the products of the differentelectrophoretic probes or e-tag probes containing polypeptide-bindingregions can be accurately detected, so as to determine the occurrence ofthe binding event involving the induced binding site. Following thebinding event, one or more reaction products are produced that exhibitmobilities different from the e-tag probe or probes from which thereaction products derive. The released form of the e-tag, termed thea-tag reporter, exhibits a different mobility and/or mass than the e-tagprobe from which it derives. The invention offers a high degree ofversatility for screening known and unknown materials. Anelectrophoretic device may be employed for separation and detection ofthe e-tag reporter. The electrophoretic device may be connected to adata processor for receiving and processing data from the device, aswell as operating the electrophoretic device.

The systems are based on having libraries available comprising aplurality of e-tag reagents that comprise at least a plurality ofdifferent mobility-modifying moieties, so as to be separable byelectrophoresis with the entities to which the mobility-modifyingmoieties are attached. The mobility-modifying moieties are retained inthe product of the reaction, where the product is modified by the gainand/or loss of a group that changes the mass and may also change thecharge of the product, as compared to the starting material. Themobility-modifying moiety is joined to a polypeptide-binding region by acleavable bond, so that the mobility-modifying moiety is released foranalysis subsequent to the binding of the induced binding site to thefirst binding agent.

As mentioned above, the present invention has application, among others,to the area of post-translational modification. One can determine theresponse of the host cell, organelles or the like to changes in thechemical and physical environments in relation to a plurality ofpathways, changes in the surface protein population, changes due toaging, neoplasia, activation, or other naturally occurring phenomenon,where the amount of protein can be quantitated. The methodologies thatmay be employed may be heterogeneous or homogeneous. Heterogeneoustechniques normally involve a separation step, where unbound label isseparated from bound label. On the other hand, homogeneous assays do notrequire, but may employ, a separation step.

In addition, in many heterogeneous assays it is required that theunbound labeled reagent be separable from the bound labeled reagent.This can be achieved in a variety of ways, each requiring a reagentbound to a solid support that distinguishes between the complex oflabeled reagent and polypeptide. The solid support may be a vessel wall,e.g., microtiter well plate well, capillary, plate, slide, beads,including magnetic beads, liposomes, or the like. The primarycharacteristics of the solid support is that it permits segregation ofthe bound labeled specific binding member from unbound probe and thatthe support does not interfere with the formation of the bindingcomplex, nor the other operations of the determination.

The solid support may have the complex directly or indirectly bound tothe support. For directly bound, one may have the first reagent or e-tagprobe covalently or non-covalently bound to the support. The surface maybe activated with various functionalities that will form covalent bondswith the first reagent. These groups may include imino halides,activated carboxyl groups, e.g., mixed anhydrides or acyl halides, aminogroups, a-halo or pseudohaloketones, etc. A specific binding memberbound to the surface of the support may be used to bind a member of thecomplex.

Usually, in a heterogeneous mode, the unbound labeled reagent or e-tagprobe will be removed by washing the bound material. Where particles orbeads are employed, these may be separated from the supernatant beforewashing, by filtration, centrifugation, magnetic separation, etc. Afterwashing, the support may be combined with a liquid into which the e-tagreporters are to be released and/or the functionality of the e-tagmoieties is reacted with the detectable label, followed by or precededby release. Depending on the nature of the cleavable bond and the methodof cleavage, the liquid may include any additional reagents for thecleavage. Where reagents for cleavage are not required, the liquid isconveniently an electrophoretic buffer. For example, where the cleavablelinkage is photolabile, the medium may be irradiated with light ofappropriate wavelength to release the a-tag reporters. Where detectablelabels are not present on the a-tag moieties, the e-tag reporters may bereacted with detectable labels. In some instances the detectable labelmaybe part of the reagent cleaving the cleavable bond, e.g., a disulfidewith a thiol. Where there is a plurality of different functionalities ondifferent binding members for reaction with the label, the differentlabels have functionalities that react with one of the functionalities.The different labels may be added together or individually in asequential manner. For example, where the functionalities involvethiols, carboxyl groups, aldehydes and olefins, the labels could haveactivated olefins, alcohols, amines and thiol groups, respectively. Byhaving removable protective groups for one or more of thefunctionalities, the protective groups may be removed stepwise and thelabels added stepwise. In this way cross-reactivity may be avoided.Whether one has the detectable label present initially or one adds thedetectable label is not critical to this invention and is frequentlygoverned by whether the polypeptide itself is cleaved by thecleavage-inducing moiety or by the nature of the polypeptides and thefirst reagent and electrophoretic probes.

In some embodiments, the e-tag reporters may be required to be separatedfrom the reagent solution, where the reagent interferes with theelectrophoretic analysis. Depending on the nature of the a-tag reportersand the reagent, one may sequester the a-tag reporters from the reagentby using ion exchange columns, liquid chromatography, an initialelectrophoretic separation, and the like. Alternatively, as discussedpreviously, one may have a capture ligand bound to the e-tag moiety orretained portion of the target-binding region for isolating the e-tagreporter, so as to remove any interfering components in the mixture.Once the solution of e-tag reporters is prepared and free of anyinterfering components, the solution may be analyzedelectrophoretically. The analysis may employ capillary electrophoresisdevices, microfluidic devices or other devices that can separate aplurality of compounds electrophoretically, providing resolved bands ofthe individual e-tag reporters.

Preferably, the assays in accordance with the present invention arecarried out in a homogeneous manner. The protocols for the subjecthomogeneous assays generally follow the procedures for the analogousheterogeneous assays. These protocols employ a signal producing systemthat includes the label of the e-tag probe, the cleavable bondassociated with the e-tag probe or the polypeptide as the case may be,electromagnetic radiation or other reagents involved in the reaction orfor diminishing background signal. In assays involving the production ofsinglet oxygen, it may be desirable to have a molecule in solution thatdegrades hydrogen peroxide to reduce its lifetime, in order to preventreaction between hydrogen peroxide produced by a bound and unboundlabel-containing reagent.

Generally, the concentrations of the various agents involved with thesignal producing system will vary with the concentration range of theindividual polypeptides in the samples to be analyzed, generally beingin the range of about 10 nM to about 10 mM. Buffers will ordinarily beemployed at a concentration in the range of about 10 to about 200 mM.The concentration of each polypeptide will generally be in the range ofabout 1 μM to about 100 μM, more usually in the range of about 100 μM toabout 10 μM. In specific situations the concentrations may be higher orlower, depending on the nature of the analyte, the affinity of thereciprocal binding members, the efficiency of release of the e-tagreporters, the sensitivity with which the e-tag reporters are detected,and the number of polypeptides, as well as other considerations.

In accordance with one aspect of the invention, a group of polypeptidesmay be monitored in a multiplexed reaction. In this case, a plurality ofpairs of a-tag probes corresponding to the various polypeptides arecombined with a sample in a single reaction vessel under conditionswhere the e-tag reporter is released from the reagent when a respectivepolypeptide binds to a binding agent and the medium is treated togenerate singlet oxygen. The e-tag reporters are either labeled fordetection or the label is added by means of a reactive functionalitypresent on the a-tag moiety. The labeled e-tag reporters of the reactionare resolved from one another on the electrophoretic device, and againare monitored as they move past the detector. The level of multiplexingpossible in this system is limited only by the degree of resolution thatcan be obtained between a designated set of a-tag reporters on theelectrophoretic device.

An additional degree of flexibility can be conferred on the assay by thestage at which the e-tag moieties are labeled. As described above, eacha-tag moiety may already contain a detectable label when introduced intothe reaction. Alternatively, an e-tag moiety may contain a functionalityallowing it to bind to a label after reaction with the sample iscomplete. In this embodiment, an e-tag probe comprising a functionalityfor binding to a detectable label is combined with a sample. After areaction to modify the mobility of the e-tag probe if its targetpolypeptide is present in the sample, additional reagents are combinedin a sample vessel with the products of the first reaction, which reactswith the modified a-tag reporter(s) to add a detectable label.

For quantitation, one may choose to use controls, which provide a signalin relation to the amount of the target that is present or isintroduced. A control to allow conversion of relative fluorescentsignals into absolute quantities is accomplished by addition of a knownquantity of a fluorophore to each sample before separation of the e-tagreporters by electrophoresis. Any fluorophore that does not interferewith detection of the e-tag reporter signals can be used for normalizingthe fluorescent signal. The control signal will preferably have anelectrophoretic mobility that is different from that of any of the a-tagreporters in the sample, and could have the same or a different emissionwavelength. Exemplary fluorescent molecules include ROX, FAM, andfluorescein.

One example of an assay in accordance with the present inventioninvolves the detection of the phosphorylation of a polypeptide. Thesample comprises cellular material and the post-translationalmodification is the phosphorylation of a particular polypeptide,referred to as a target polypeptide. The sample is combined with a firstreagent comprising a photosensitizer linked to a metal affinity agent towhich is bound a metal ion. If the phosphorylated target polypeptide ispresent, the phosphate group binds to the metal-metal affinity agentcomplex. A electrophoretic probe is combined with the above reactionmixture. The electrophoretic probe comprises an antibody for the targetpolypeptide, to which is cleavably linked an e-tag reporter. Thecleavable link comprises a moiety that is cleavable by singlet oxygen.After addition of the electrophoretic probe and an appropriateincubation period, the reaction mixture is irradiated with light toexcite the photosensitizes, which generates singlet oxygen. Thecleavable moiety is cleaved by the singlet oxygen because the cleavablemoiety is in close proximity to the photosensitizer and the activespecies, namely, singlet oxygen, retains sufficient activity to cleavethe cleavable moiety and release a-tag reporter. Electrophoretic probethat does not become bound to target polypeptide because the targetpolypeptide is not present, or excess electrophoretic probe, orelectrophoretic probe that binds to a polypeptide that is notphosphorylated, does not yield cleaved a-tag reporters because theactivity of the singlet oxygen is very short-lived and the cleavablemoiety in any electrophoretic probe that is not bound to the firstreagent by virtue of the presence of phosphorylated target polypeptidedoes not yield cleaved e-tag reporters. The released e-tag reporter isseparated on the basis of its different mobility and detected on thebasis of the detection moiety that remains attached to the mobilitymodifying moiety of the a-tag reporter. The presence and/or amount ofthe released e-tag reporter indicates the presence and/or amount of thetarget polypeptide.

The present invention finds particular use in multiplexed assays fortarget polypeptides. An example of an assay in accordance with thisaspect of the present invention involves the detection of thephosphorylation of multiple polypeptides. The sample comprises cellularmaterial and the post-translational modification is the phosphorylationof several polypeptides, referred to as target polypeptides. The sampleis combined with a first reagent comprising a photosensitizer linked toa metal affinity agent to which is bound a metal ion. The first reagentis a class-specific reagent in that it binds to any phosphate grouppresent in the reaction mixture. If the phosphorylated targetpolypeptides are present, the phosphate group binds to the metal-metalaffinity agent complex. A plurality of electrophoretic probes iscombined with the above reaction mixture. Each of the electrophoreticprobes comprises an antibody for a particular target polypeptide, towhich is cleavably linked an e-tag reporter that is unique for theparticular target polypeptide. The cleavable link comprises a moietythat is cleavable by singlet oxygen. After addition of theelectrophoretic probes and an appropriate incubation period, thereaction mixture is irradiated with light to excite the photosensitizer,which generates singlet oxygen. The cleavable moiety is cleaved by thesinglet oxygen because the cleavable moiety is in close proximity to thephotosensitizer and the active species, namely, singlet oxygen, retainssufficient activity to cleave the cleavable moiety and release e-tagreporters from all electrophoretic probes that are bound to a targetpolypeptide bound to the class-specific reagent. Again, electrophoreticprobes, which do not become bound to target polypeptides bound to theclass-specific reagent, do not yield cleaved e-tag reporters for thereasons given above. The released a-tag reporters are separated on thebasis of their differences in mobility and detected on the basis of thedetection moiety that remains attached to the mobility modifying moietyof the a-tag reporter. The presence and/or amount of each of thereleased a-tag reporters indicate the presence and/or amount of each ofthe respective target polypeptides. In this fashion various cellularpathways may be studied on a real time basis. Protein phosphorylationand de-phosphorylation reactions may be studied to develop moreinformation about metabolic regulation and signal transduction pathways.The above method may be repeated at various times during the cell cycleto follow the progression of the cell.

Another application of the present invention is to detect multiplephosphorylations of a target polypeptide. For example, it is desirableto know whether a polypeptide has been mono-phosphorylated,bis-phosphorylated or even higher multiples of phosphorylation. Anexample of an assay in accordance with this aspect of the presentinvention involves the detection of the degree of phosphorylation of atarget polypeptide. The sample, which comprises cellular material, iscombined with a first reagent comprising a multiple photosensitizermolecules linked to a hub molecule to which multiple molecules of ametal affinity agent with bound metal are also linked. By appropriatetitration of the class-specific reagent, the level of phosphorylation ofthe target polypeptide can be determined. If the phosphorylated targetpolypeptides are present, the phosphate group binds to the metal-metalaffinity agent complex. An electrophoretic probe is combined with theabove reaction mixture. The electrophoretic probe comprises an antibodyfor the particular target polypeptide, to which is cleavably linked ane-tag reporter that is unique for the particular target polypeptide. Thecleavable link comprises a moiety that is cleavable by singlet oxygen.After addition of the electrophoretic probe and an appropriateincubation period, the reaction mixture is irradiated with light toexcite the photosensitizer, which generates singlet oxygen. Thecleavable moiety is cleaved by the singlet oxygen because the cleavablemoiety is in close proximity to the photosensitizer. The active species,namely, singlet oxygen, retains sufficient activity to cleave thecleavable moiety and release e-tag reporters from the electrophoreticprobe that is bound to a target polypeptide bound to the class-specificreagent. Again, electrophoretic probes, which do not become bound totarget polypeptides bound to the class-specific reagent, do not yieldcleaved e-tag reporters for the reasons given above. The released e-tagreporter is separated on the basis of differences in mobility anddetected on the basis of the detection moiety that remains attached tothe mobility modifying moiety of the e-tag reporter. The presence and/oramount of the released e-tag reporter may be correlated with the amountof class-specific reagent added to determine the level ofphosphorylation of the target polypeptide.

The present invention may be employed to determine the site or sites ofphosphorylation on a target polypeptide. In an example of an assay inaccordance with this aspect of the present invention, the sample, whichcomprises cellular material, is combined with a first reagent comprisinga chemical protease linked to a metal affinity agent to which is bound ametal ion. If the phosphorylated target polypeptide is present, thephosphate group binds to the metal-metal affinity agent complex. Thechemical protease is activated by irradiation with light and sitespecific cleavage takes place on the target polypeptide whose phosphategroup is bound to the metal affinity-metal complex. The cleavageproducts represent unique moieties, or e-tag moieties, which may beanalyzed directly by, for example, electroseparation. On the other hand,one or more electrophoretic probes may be combined with the abovereaction mixture to provide a detection moiety for the unique moieties.Each electrophoretic probe comprises an antibody for a cleaved moiety,to which is attached the detection moiety. The e-tag reporter and isseparated on the basis of its different mobility and detected on thebasis of the detection moiety that is attached. The presence of thea-tag reporter is indicative of the site of phosphorylation of thetarget polypeptide.

Another example of an assay in accordance with the present inventioninvolves the detection of the glycosylation of multiple polypeptides.The sample comprises cellular material and the post-translationalmodification is the glycosylation of several polypeptides, referred toas target polypeptides. The sample is combined with a first reagentcomprising a photosensitizer linked to a boronic acid containing agent.The first reagent is a class-specific reagent in that it binds to anycarbohydrate moiety present in the reaction mixture. If the glycosylatedtarget polypeptides are present, the carbohydrate group binds to theboronic acid containing agent. A plurality of electrophoretic probes iscombined with the above reaction mixture. Each of the electrophoreticprobes comprises an antibody for a particular target polypeptide, towhich is cleavably linked an a-tag reporter that is unique for theparticular target polypeptide. The cleavable link comprises a moietythat is cleavable by singlet oxygen. After addition of theelectrophoretic probes and an appropriate incubation period, thereaction mixture is irradiated with light to excite the photosensitizer,which generates singlet oxygen. The cleavable moiety is cleaved by thesinglet oxygen thereby releasing a-tag reporters from allelectrophoretic probes that are bound to a target polypeptide bound tothe class-specific reagent. Again, electrophoretic probes, which do notbecome bound to target polypeptides bound to the class-specific reagent,do not yield cleaved a-tag reporters for the reasons given above. Thereleased e-tag reporters are separated on the basis of their differencesin mobility and detected on the basis of the detection moiety thatremains attached to the mobility modifying moiety of the e-tag reporter.The presence and/or amount of each of the released a-tag reportersindicates the presence and/or amount of each of the respective targetpolypeptides, i.e., the glycosylated polypeptides. The above method maybe repeated at various times during the cell cycle to follow theprogression of the cell.

The present invention has broad application to the study of cellularsignaling pathways including, by way of illustration and not limitation,MAP kinase pathways, the Ras/ERK MAPK pathway, the JNK/SAPK and otherMAPK pathways, JAK/STAT pathways, NF-OB and dorsal, NF-AT dual signalingpathway, regulation of lymphocyte function, T cell antigen receptorsignal transduction, various signal transducers and activators oftranscription, cell division cycle check points, and the like.

Mitogen-activated protein kinases (MAPK's) may provide and understandingof cellular events in growth factor and cytokine receptor signaling. TheMAP kinases (also referred to as extracellular signal-regulated proteinkinases, or ERK's) are the terminal enzymes in a three-kinase cascade.The reiteration of three-kinase cascades for related but distinctsignaling pathways gave rise to the concept of a MAPK pathway as amodular, multifunctional signaling element that acts sequentially withinone pathway, where each enzyme phosphorylates and thereby activates thenext member in the sequence. The recent identification of distinct MAPKcascades that are conserved across all eukaryotes indicates that theMAPK module has been adapted for interpretation of a diverse array ofextracellular signals. The MAPK superfamily of enzymes is a criticalcomponent of a central switchboard that coordinates incoming signalsgenerated by a variety of extracellular and intracellular mediators.Specific phosphorylation and activation of enzymes in the MAPK moduletransmits the signal down the cascade, resulting in phosphorylation ofmany proteins with substantial regulatory functions throughout the cell,including other protein kinases, transcription factors, cytoskeletalproteins and other enzymes. (Cobb, et al., Promega Notes Magazine (1996)59:37, et seq.)

Another class of assays involves the association of a photosensitizerreagent with a cell to study, for example, post-translationalmodifications, small molecules that alter and/or control the functionand/or expression of proteins to which they bind, identification ofnovel proteins using known small molecules, screening of characterizedtargets, such as kinases, peptides, etc., screens for functional classesof proteins to identify novel targets, such as, e.g., kinases,phosphatases, DNA- or RNA-binding proteins, peptides, proteases, etc.,and so forth. For example, a small molecule thought to bind to thetarget of interest can be conjugated to a photosensitizer molecule,forming a small molecule-sensitizer complex. Antibody molecules that canbind various targets of interest will be labeled with different e-tagmoieties, forming an e-tag-antibody complex. The presence or absence ofthe particular target can then be investigated and quantitated byforming the e-tag-antibody::target::small molecule-sensitizer complex.Association of the antibody and small molecule with the target willbring the e-tag moiety and photosensitizer in close proximity, enablingrelease of the e-tag reporter. The photosensitizer may be attached to asmall molecule, peptide, inhibitor, lipid, carbohydrate, an antibodymolecule, oligonucleotide, and the like. The photosensitizer may also beattached to the surface of microorganisms or cells that carry any of theabove structures, allowing one to monitor interactions between cellsurface molecules of two separate cells. For cell surface studies, thephotosensitizer reagent may be associated with the cell by attachment toa specific cell surface moiety, or non-specific incorporation into thecell membrane in a manner in which the photosensitizer is free todiffuse within the membrane.

A particular assay for kinase interactions is illustrative of the manytypes of assays that can be developed. Specific kinase interactions maybe studied using a sandwich assay format. For example, a firstanti-kinase antibody is labeled with an e-tag moiety, and a secondantibody that binds a phosphorylated molecule, will be labeled with aphotosensitizer. After forming the immune complex, the e-tag moiety willbe released by illuminating with light at the appropriate wavelength,then separated by capillary electrophoresis. In another format, kinaseswith varying specificities can be investigated. Anti-kinase antibodiesare labeled with the same photosensitizer (e.g.,photosensitizer₁-anti-kinase₁, photosensitizer₁-anti-kinase₂,photosensitizers-anti-kinase₃, etc.), while specific kinase substratesor inhibitors will be labeled with specific e-tag moieties (e.g.,substrate₁-e-tag₁, substrate₂-e-tag₂, substrate₃-e-tag₃, etc.). Bindingcomplexes will be formed, either stably or transiently. Afterillumination and electrophoretic separation, particular patterns will begenerated that are indicative of the various levels of the chosen kinasetargets in the sample, determined simultaneously. Once a signal patternis established, the effects of various drugs, treatments, or geneticalterations can be assessed as alterations to this pattern.

An example of use of the present invention for proteomics is given inFIG. 7. In this embodiment, assays are performed by first lysing a cellsample. All the proteins in the cell lysate are labeled with acleavage-inducing moiety. The labeling is performed to obtain at leastone cleavage-inducing moiety per protein. As shown in FIG. 7, theproteins are interrogated with anti-protein ligand (APL), which can be areceptor or an antibody. The APL's are labeled with specific eTagreporter molecules through a cleavable linkage; each ligand has a uniqueeTag reporter associated with it. After binding, the cleavage-inducingmoieties are activated to release the different reporters (Y). Thecleavage-inducing moiety and the cleavable linkage must to be in closeproximity for the e-tag reporter to be released. The reporters are thenseparated via capillary electrophoresis and quantified. The technologyenables multiplexed profiling of proteins, small molecules,enzyme-substrates and cell surface receptors in a homogeneous assayformat.

Determining the cellular function of a protein generally requires ameans to alter the function. One approach is a genetic one that involvesthe use of inactivating or activating mutations in genes encoding theprotein. Inactivation involves deletion or knock-out approaches, andactivation or over-expression involves oncogenic approaches. Anotherapproach involves the use of small molecules that alter the function ofproteins to which they bind. Ligands exist that are capable of eitherinactivating or activating protein function. It has been demonstratedthat for some natural products, specificity can approach that of a geneknock-out (Schreiber, Bioorganic and Medicinal Chemistry, 6 (1998)1127-1152; Stockwell, et al., Chemistry and Biology, 6 (1999) 71-83).Small molecule natural products have aided in disentangling the complexweb of cell-cycle events in several ways such as, for example, arrestsat specific points in the cell cycle allowing synchronization of apopulation of cells. Once a specific binding interaction is established,a cell-permeable natural product can be used to understand the functionof its protein target in living cells. Some natural products inhibit thesignal transduction pathway required for the transition from thequiescent state to the G1 transition state. Small molecules can affectpost-translational events such as protein glycosylation, methylation,lipidation, isoprenylation, ubiquitination, phosphorylation andacetylation.

Entrance into and exit out of the cell cycle occurs as a cell passesbetween active proliferation and a quiescent state or G0 state, in whichthe fundamental metabolism of the cell is depressed, including many ofits usually active functions such as transcription and proteinsynthesis. Deprivation of growth factors can cause a cell to exit intoquiescent state, whereas stimulation with growth factors can signal acell to re-enter the active cycle. A cell may also exit the cell cycleto undergo processes of differentiation or programmed cell death(apoptosis). The element responsible for driving the cell cycle from onephase to the next is a series of protein kinases and phosphatases thatactivate and deactivate each other. The cyclin-dependent kinases areresponsible for phosphorylating various substrates critical tocell-cycle progression. The levels of the cyclin-dependent kinases areinvariant throughout the cell cycle, but their activities are modulatedby their interaction with another set of proteins called cyclins, whoselevels fluctuate. In addition many of the receptors on the plasmamembrane of the cell are tyrosine kinases that, upon activation,initiate an intracellular signal transduction pathway whose ultimate endpoint is cell proliferation.

The methods and reagents of the present invention are well suited forthe aforementioned area of analysis. A wide variety of small moleculescause a loss of function of their cognate targets, including kinases,phosphatases, membrane receptors, proteases, isoprenyl transferases andpolymerises. As a result of the present invention, protein function maybe studied in its intracellular environment. Proteins of interest may bequantitated. The effect on the levels of downstream proteins such as,e.g., cyclins, may be studied using the reagents of the presentinvention. Active and inactive cyclin-dependent kinases (Cdk's) may bemeasured along with the expression of cyclins. The Cdk-cyclin complexmay also be quantitated. Signal transduction pathways may be studiedwith the present reagents.

The G1 phase is a critical point at which the cell assesses whether itshould enter another full round of division. Proteins in GI progressionare frequently mutated in human cancers and are attractive targets fortherapeutic agents. It is desirable to look only at the early GI phaseor the late G1 phase to ascertain compounds that may be therapeuticagents. Currently, the approach is to look only at cell proliferation orcell death in assessing compounds. The various phases involved aredepicted in FIG. 8.

In one exemplary embodiment, cells may be grown on the bottom of asuitable container such as a well. After the cells are fixed, they maybe probed for a particular antigen by a screening method in accordancewith the present invention. A photosensitizer reagent may be employedthat is incorporated into the cell membrane or, alternatively, thephotosensitizer reagent comprises a photosensitizer attached to anantibody for the antigen, or for a class of protein on the surface ofthe cell. An e-tag reagent may be used wherein an antibody for theantigen is linked by a cleavable linkage to an a-tag moiety. Thephotosensitizer reagent is added to the fixed cells in a suitable mediumunder conditions for incorporation into the cell membrane or binding tothe antigen if present. Then, the e-tag probe is added and the mediumtreated under conditions for binding of the antibody for the antigen tothe antigen, if present. Next, the photosensitizer is activated such asby irradiation with light to generate, for example, singlet oxygen,which cleaves the cleavable linkage in the a-tag probe only if theantigen is present on the cell to bring the cleavable linkage intoproximity to the photosensitizer in the cell membrane. The medium isthen examined for the released a-tag reporter and the presence thereofindicates the presence of the antigen. Multiple antigens may be screenedat the same time by employing multiple a-tag probes, each with anantibody specific for a particular antigen linked by the cleavablelinkage to an e-tag moiety that comprises a mobility-modifying moietythat differentiates the a-tag reporter from other a-tag reportersgenerated by the possible presence of other antigens. After the abovesteps, the medium is subjected to a separation step in which the e-tagreporters are separated on the basis of their respective differences inmobility. In this way one may study protein expression by the cells.

Hypothetical results of assays conducted in a manner as discussed aboveare illustrated in FIGS. 9A, 9B and 9C. These are conceptual cell-basedassays for simultaneous monitoring of the levels of Cdk's (kinaseactivation using Olomoucine labeled release-inducing agent, e.g.,photosensitizer) and cyclins A-E. The electrophoretic probe reagentswould employ an antibody specific for one of the expressed proteins, inwhich one or more unique a-tags are releasably linked to each antibody.Referring to FIG. 9A, for control cells, i.e., cells in the absence ofcompound to be tested, eight peaks may be obtained representing Cdk2,Cdk6, Cdk4, Cdc2 and Cyclins A, D, E and B. Where a compound beingtested results in early G1 arrest (FIG. 9B), three peaks might beobserved, namely, Cdk6, Cdk4 and Cyclin D. Where a compound being testedresults in late G1 arrest (FIG. 9C), five peaks might be observed,namely, Cdk2, Cdk6, Cdk4, Cyclin D and Cyclin E. In both compoundtreatments, the compound tested may be an effective drug for treatmentof a patient.

Another assay involves the study of post-translational regulation ofproteins using a library of small molecules for regulators of reversiblecovalent modification of proteins. A set of e-tag probes is preparedwherein each e-tag probe comprises an antibody for the protein in itsmodified or unmodified state, which cleavably linked to an a-tag moietythat is unique within the set of probes. A photosensitizer reagent isincorporated into the cell, either in the membrane, the cytosol, orother appropriate location. The probes are then combined with the cellsin suitable reaction containers. Each of the small molecules of thelibrary is added to a respective container. The medium is thenirradiated as described above. The medium from each of the containers istreated to separate and identify the e-tag reporters. The presence ofone or more of the a-tag reporters is related to ability of a respectivesmall molecule to regulate the covalent modification of the proteins.

In another approach for studying post-translational modification ofproteins, a set of e-tag probes is prepared in which one of the smallmolecules of the library is cleavably linked to an e-tag moiety for eacha-tag probe. The cells in suitable containers are treated with thephotosensitizer reagent to incorporate the photosensitizer into theappropriate cellular location. The entire set of a-tag probes, orportions thereof, is added to the container under conditions wherein thesmall molecules are allowed to bring about the protein modification ofinterest. Only those small molecules that are involved in themodification of interest result in the bringing of the cleavable linkageinto close proximity to the photosensitizer. The medium from thecontainer is treated to separate and identify the a-tag reporters. Thepresence of one or more of the a-tag reporters is related to ability ofa respective small molecule to regulate the covalent modification of theprotein.

In the above manner, protein pathways may be studied in the context of acascade of cellular events that are altered by a small molecule thatacts as an inhibitor, an activator, a potential drug, hormone, enzymecofactor, or other type of regulatory factor. The influence of smallmolecules on gene expression and protein function may be studied so thatsmall molecules may be used to identify novel proteins. The use of smallmolecules may be as specific as gene knockouts. The invention may beused in the discovery of small molecules that immediately alterfunction, which is not possible in classical genetics. The smallmolecules may be employed to control the function of proteins and inscreening assays such a kinase screening, peptide screening, randomscreens such as for kinases, peptides, proteases, GPCR, and so forth.Screening assays for small molecule suppressors of cell-cycle arrestingagents may be carried out using the present reagents.

Another method of use of electrophoretic probes in accordance with thepresent invention is depicted in FIG. 11. A cell membrane is shownhaving multiple receptors including receptor R on its surface. Theantigen for specifically binding to receptor R is added and, if receptorR is present, the antigen binds to it as well as cofactor Co. Enzyme Eabinds to the above complex, which then activates Ea to convert P1 to itsphosphorylated counterpart P1a. Kinase P2 binds to P1a to form an activecomplex. This system may be used to screen for inhibitors, such as,e.g., small molecules or drugs, of the activated complex. Accordingly,the reagents for this assay include each molecule to be screened as aninhibitor where each molecule is bound to a release-inducing reagent.The electrophoretic probe is antibody (Ab) to the complex, e.g., to theP1a of the complex, where the antibody is releasably linked to multipleelectrophoretic moieties. If one of the molecules being screened bindsto and inhibits the activity of the complex, the release-inducingreagent is brought into proximity of the electrophoretic probes and thee-tag moieties are released, detected and/or quantitated.

The above approach may be employed also to screen proteins for bindingability to a particular receptor. In this approach consistent with thescheme of FIG. 11, drugs that have well-defined targets may be employed.Only if one or more of such targets are present by virtue of the proteinbinding to receptor R, will e-tag reporters be detected. As can be seen,the methods of the invention are versatile in which entity is the knownand which is the unknown.

A variation of the scheme of FIG. 11 is depicted in FIG. 12. In thisembodiment the release-inducing reagent is an antibody Ab1, whichspecifically binds to the active complex of P2a and P1a. Theelectrophoretic probe is antibody Ab2 to which multiple e-tag moietiesare releasably linked. Ligands L may be studied for their ability tobind to receptor R on the surface of a cell. If L does bind to R, theactive complex of P2a and P1a will form. Antibodies Ab1 and Ab2 bind tothe active complex, bringing the releasable linkage into close proximitywith the release-inducing reagent. The e-tag reporters are released,detected and quantitated.

FIG. 13A depicts a hypothetical example of a protein-protein interactionpathway involving multiple phosphorylation events, to which the presentmethods and compositions may be applied. In this illustration,protein-protein binding is promoted by small molecules. FRAP resultsfrom the interaction of nutrients and mitogens with receptors on thesurface of the cell. As can be seen in FIG. 13A, FKBP proteins bind toFRAP (binding event 1) in the presence of rapamycin resulting in 4E-BP1, which is involved in control of mRNA translation initiation. PP2A,which is a kinase activated by FRAP (binding event 2), yields p70S6K inthe presence of calyculin-A, which leads to S6 protein binding (bindingevent 5). This latter protein is also involved in control of mRNAtranslation initiation. Another receptor, RTK, on the surface of a cellis acted upon by growth factor to produce kinase PI3K. In the presenceof Wortmannin, PI3K is converted to PDK1 (binding event 6), which yieldsp70S6K leading to S6 protein. PDK1 also yields PKB (binding event 3),which in turn yields FRAP (binding event 4). The graphs in FIG. 13B showthe results of assays wherein no drug is present or wherein eitherWortmannin, Rapamycin or Calyculin is present, each conjugated to arelease-inducing reagent and a electrophoretic probe is employedcomprising an antibody for one of the proteins involved in a bindingevent releasably linked to an electrophoretic moiety. The pathway abovecan assist in looking at the various binding events involved, atinhibitors of one or more of these binding events, at competitors in oneor more of the binding events, at modulators of kinase activity, and soforth. With the methods and reagents of the invention one or more of theabove may be monitored simultaneously in a single assay using multipleelectrophoretic probes.

Analysis of Reaction Products

Methods for electrophoresis of are well known and are described, forexample, in Krylov et al, Anal. Chem., 72: 111R-128R (2000); P. D.Grossman and J. C. Colburn, Capillary Electrophoresis: Theory andPractice, Academic Press, Inc., NY (1992); U.S. Pat. Nos. 5,374,527;5,624,800; 5,552,028; ABI PRISM 377 DNA Sequencer User's Manual, Rev. A,January 1995, Chapter 2 (Applied Biosystems, Foster City, Calif.); andthe like. A variety of suitable electrophoresis media are commerciallyavailable from Applied Biosystems and other vendors, includingnon-crosslinked media, for use with automated instruments such as theApplied Biosystems “3700” and “3100” Instruments, for example. Optimalelectrophoresis conditions, e.g., polymer concentration, pH,temperature, voltage, concentration of denaturing agent, employed in aparticular separation depends on many factors, including the size rangeof the compounds to be separated, their compositions, and the like.Accordingly application of the invention may require standardpreliminary testing to optimize conditions for particular separations.

During or after electrophoretic separation, the electrophoretic tags aredetected or identified by recording fluorescence signals and migrationtimes (or migration distances) of the separated compounds, or byconstructing a chart of relative fluorescent and order of migration ofthe electrophoretic tags (e.g., as an electropherogram). To perform suchdetection, the electrophoretic tags can be illuminated by standardmeans, e.g. a high intensity mercury vapor lamp, a laser, or the like.Typically, the electrophoretic tags are illuminated by laser lightgenerated by a He—Ne gas laser or a solid-state diode laser. Thefluorescence signals can then be detected by a light-sensitive detector,e.g., a photomultiplier tube, a charged-coupled device, or the like.Exemplary electrophoresis detection systems are described elsewhere,e.g., U.S. Pat. Nos. 5,543,026; 5,274,240; 4,879,012; 5,091,652;6,142,162; or the like.

After completion of the reaction, which may be monitored, for example,by monitoring the change in signal such as, e.g., fluorescence asdescribed above, or taking aliquots and assaying for total free e-tagreporters, the mixture may now be analyzed. Depending on the instrument,from one to four different fluorescers activated by the same lightsource and emitting at different detectable labels may be used. Withimprovements, five or more different fluorescers may be available, wherean additional light source may be required. Electrochemical detection isdescribed in U.S. Pat. No. 6,045,676.

In one embodiment of the presence of each of the cleaved e-tag reportersis determined by the fluorescent label contained in the e-tag moiety.The separation of the mixture of labeled e-tag reporters is typicallycarried out by electroseparation, which involves the separation ofcomponents in a liquid by application of an electric field, preferably,by electrokinesis (electrokinetic flow) or electrophoretic flow, or acombination of electrophoretic flow within electroosmotic flow, with theseparation of the e-tag reporter mixture into individual fractions orbands. Electroseparation involves the migration and separation ofmolecules in an electric field based on differences in mobility. Variousforms of electroseparation include, by way of example and notlimitation, free zone electrophoresis, gel electrophoresis, isoelectricfocusing, isotachophoresis, capillary electrochromatography, andmicellar electrokinetic chromatography. Capillary electrophoresisinvolves electroseparation, preferably by electrokinetic flow, includingelectrophoretic, dielectrophoretic and/or electroosmotic flow, conductedin a tube or channel of about 1 to about 200 micrometer, usually, about10 to about 100 micrometers cross-sectional dimensions. The capillarymay be a long independent capillary tube or a channel in a wafer or filmcomprised of silicon, quartz, glass or plastic.

In capillary electroseparation, an aliquot of the reaction mixturecontaining the e-tag reporters is subjected to electroseparation byintroducing the aliquot into an electroseparation channel that may bepart of, or linked to, a capillary device in which the amplification andother reactions are performed. An electric potential is then applied tothe electrically conductive medium contained within the channel toeffectuate migration of the components within the combination.Generally, the electric potential applied is sufficient to achieveelectroseparation of the desired components according to practices wellknown in the art. One skilled in the art will be capable of determiningthe suitable electric potentials for a given set of reagents used in thepresent invention and/or the nature of the cleaved labels, the nature ofthe reaction medium and so forth. The parameters for theelectroseparation including those for the medium and the electricpotential are usually optimized to achieve maximum separation of thedesired components. This may be achieved empirically and is well withinthe purview of the skilled artisan.

For a homogeneous assay, the sample, the first and electrophoreticprobes, and ancillary reagents are combined in a reaction mixturesupporting the cleavage of the linking region. The mixture may beprocessed to separate the e-tag reporters from the other components ofthe mixture. The mixture, with or without a-tag reporter enrichment, maythen be transferred to an electrophoresis device, usually a microfluidicor capillary electrophoresis device and the medium modified as requiredfor the electrophoretic separation. Where one wishes to remove from theseparation channel intact a-tag reporter molecules, a ligand is bound tothe a-tag reporter that is not released when the a-tag reporter isreleased. Alternatively, by adding a reciprocal binding member that hasthe opposite charge of the a-tag reporter, so that the overall charge isopposite to the charge of the a-tag reporter, these molecules willmigrate toward the opposite electrode from the released a-tag reportermolecules. For example, one could use biotin and streptavidin, wherestreptavidin carries a positive charge. In the case of a peptideanalyte, one embodiment would have cleavage at a site where the ligandremains with the peptide analyte. For example, one could have the a-tagmoiety substituted for the methyl group of methionine. Using thepyrazolone of the modified methionine, one could bond to an availablelysine. The amino group of the pyrazolone would be substituted withbiotin. Cleavage would then be achieved with cyanogen bromide, releasingthe a-tag reporter, but the biotin would remain with the peptide and 0any e-tag moiety that was not released from the binding member. Avidinis then used to change the polarity or sequester the e-tag moietyconjugated to the target-binding moiety for the analyte ortarget-binding moiety.

For capillary electrophoresis one may employ one or more detection zonesto detect the separated cleaved labels. It is, of course, within thepurview of the present invention to utilize several detection zonesdepending on the nature of the reactions, mobility-modifying moieties,and so forth. There may be any number of detection zones associated witha single channel or with multiple channels. Suitable detectors for usein the detection zones include, by way of example, photomultipliertubes, photodiodes, photodiode arrays, avalanche photodiodes, linear andarray charge coupled device (CCD) chips, CCD camera modules,spectrofluorometers, and the like. Excitation sources include, forexample, filtered lamps, LEDs, laser diodes, gas, liquid and solid-statelasers, and so forth. The detection may be laser scanned excitation, CCDcamera detection, coaxial fiber optics, confocal back or forwardfluorescence detection in single or array configurations, and the like.

Detection may be by any of the known methods associated with theanalysis of capillary 35 electrophoresis columns including the methodsshown in U.S. Pat. No. 5,560,811 (column 11, lines 19-30), U.S. Pat.Nos. 4,675,300, 4,274,240 and 5,324,401, the relevant disclosures ofwhich are incorporated herein by reference. Those skilled in theelectrophoresis arts will recognize a wide range of electric potentialsor field strengths may be used, for example, fields of 10 to 1000 V/cmare used with about 200 to about 600 V/cm being more typical. The uppervoltage limit for commercial systems is about 30 kV, with a capillarylength of about 40 to about 60 cm, giving a maximum field of about 600V/cm. For DNA, typically the capillary is coated to reduceelectroosmotic flow, and the injection end of the capillary ismaintained at a negative potential.

For ease of detection, the entire apparatus may be fabricated from aplastic material that is optically transparent, which generally allowslight of wavelengths ranging from about 180 to about 1500 nm, usuallyabout 220 to about 800 nm, more usually about 450 to about 700 nm, tohave low transmission losses. Suitable materials include fused silica,plastics, quartz, glass, and so forth.

Kits for Use of the e-tag Reagents

As a matter of convenience, predetermined amounts of reagents employedin the present invention can be provided in a kit in packagedcombination. One exemplary kit for polypeptide analysis can comprise inpackaged combination a first reagent comprising a cleavage-inducingmoiety and a binding agent for binding to a binding site on thepolypeptide that has undergone a post-translational modification. Thekit can further comprise one or more electrophoretic probes comprising aspecific binding agent for a particular polypeptide cleavably linked toan e-tag reporter. For example, each of the e-tag probes may comprise apolypeptide-binding moiety such as an antibody cleavably linked to ane-tag moiety. The mobility-modifying moiety of each of the e-tag probeshas a mobility that allows differentiation of one a-tag reporter fromanother and is unique to a particular protein of interest. The kits willinclude at least about 1, usually at least about 10, more usually atleast about 20 and frequently at least about 50 or more different probesthat can generate e-tag reporters that can be separated by theirmobility. On the other hand, where the polypeptide itself isspecifically cleaved to provide an e-tag moiety, the kit may includereagents wherein each reagent comprises a detection moiety linked to amoiety for binding to a specific cleaved e-tag moiety.

The kit may further comprise a device for conducting capillaryelectrophoresis as well as reagents that may be necessary to activatethe cleavage-inducing moiety of the cleavage-inducing reagent. The kitcan further include various buffered media, some of which may containone or more of the above reagents.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents necessary toachieve the objects of the present invention. Under appropriatecircumstances one or more of the reagents in the kit can be provided asa dry powder, usually lyophilized, including excipients, which ondissolution will provide for a reagent solution having the appropriateconcentrations for performing a method or assay in accordance with thepresent invention. Each reagent can be packaged in separate containersor some reagents can be combined in one container where cross-reactivityand shelf life permit. The kits may also include a written descriptionof a method in accordance with the present invention as described above.

EXAMPLES

The invention is demonstrated further by the following syntheses andillustrative examples. Parts and percentages are by weight unlessotherwise indicated. Temperatures are in degrees Centigrade (° C.)unless otherwise specified. The following preparations and examplesillustrate the invention but are not intended to limit its scope. Unlessotherwise indicated, peptides used in the following examples wereprepared by synthesis using an automated synthesizer and were purifiedby gel electrophoresis or HPLC.

The following abbreviations have the meanings set forth below:

Tris HCl—Tris(hydroxymethyl)aminomethane-HCI (a 10× solution) fromBioWhittaker, 20 Walkersville, Md.

HPLC—high performance liquid chromatography

TLC—thin layer chromatography

BSA—bovine serum albumin, e.g. available from Sigma Chemical Company(St. Louis, Mo.), or like reagent supplier.

EDTA—ethylene diamine tetra-acetate from Sigma Chemical Company

g—grams

mM—millimolar

FAM—carboxyfluorescein

EMCS—N-ε-maleimidocaproyloxy-succinimide ester

EDC—1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

NHS—N-hydroxysuccinimide

DCC—1,3-dicylcohexylcarbodiimide

DMF—dimethylformamide

Fmoc—N-(9-fluorenylmethoxycarbonyl)-

Example 1 Conjugation of Photosensitizer Molecules to Assay Reagents

Photosensitizer molecules are conjugated to a metal affinity agent, aboronic acid containing agent, a hub molecule, and the like by variousconventional methods and configurations. For example, an activated (NHSester, aldehyde, sulfonyl chloride, etc) photosensitizer (Rose Bengal,phthalocyanine, etc.) can be reacted with reactive amino-groupcontaining moieties (aminodextran, amino-group containing agents (withappropriate protection of metal binding sites), other small and largemolecules). The formed conjugates can be used directly (for example theantibody-photosensitizer conjugate, Biotin-LC-photosensitizer, etc.) invarious assays. Also, the formed conjugates can be further coupled withantibody (for example, aminodextran-photosensitizer conjugate containing20-200 photosensitizers and 200-500 amino-groups can be coupled toperiodate oxidized antibody molecules to generate theantibody-dextran-sensitizer conjugate) or with the antibody and aparticle. For example, aminodextran-sensitizer conjugate containing20-200 photosensitizers and 200-500 amino-groups can be coupled tocarboxylated polystyrene beads by EDC coupling chemistry to form thephotosensitizer-aminodextran-particle conjugate. Methods forincorporation of a photosensitizer into a particle are given in, e.g.,U.S. Pat. No. 5,340,716. Then the Na-periodate oxidized antibodymolecules can be reacted with the amino-groups of the aminodextranmolecule, in presence of sodium cyanoborohydride, to generate theantibody-dextran-photosensitizer-particle conjugate, referred to hereinas a “photosensitizer bead.” It should be noted that instead of anantibody molecule, avidin or other molecules can be used also.

Example 2 Conjugation of an a-Tag Moiety and Release of an e-TagReporter

FIG. 14 summarizes the methodology for conjugation of an e-tag moiety toan antibody or other binding moiety with a free amino group, and thereaction of the resulting conjugate with singlet oxygen to produce asulfinic acid moiety as the released e-tag reporter. FIG. 15 A-J showsseveral a-tag reagents, most of which utilize 5- or 6-carboxyfluorescein(FAM) as starting material.

Example 3 Preparation of Pro2, Pro4, and Pro6 Through Pro13

The scheme outlined in FIG. 16A shows a five-step procedure for thepreparation of the carboxyfluorescein-derived a-tag moieties, namely,Pro2, Pro4, Pro6, Pro7, Pro8, Pro9, Pro10, Pro11, Pro12, and Pro13. Thefirst step involves the reaction of a 5- or 6-FAM withN-hydroxysuccinimide (NHS) and 1,3-dicylcohexylcarbodiimide (DCC) in DMFto give the corresponding ester, which was then treated with a varietyof diamines to yield the desired amide, compound 1. Treatment ofcompound 1 with N-succinimidyl iodoacetate provided the expectediodoacetamide derivative, which was not isolated but was further reactedwith 3-mercaptopropionic acid in the presence of triethylamine. Finally,the resulting β-thioacid (compound 2) was converted, as described above,to its NHS ester. The various e-tag moieties were synthesized startingwith 5- or 6-FAM, and one of various diamines. The diamine is givenH₂N^X^NH₂ in the first reaction of FIG. 16A. The regioisomer of FAM andthe chemical entity of “X” within the diamine are indicated in the tablebelow for each of the e-tag moieties synthesized. Clearly, the diamine,X, can have a wide range of additional forms, as described above in thediscussion of the mobility modifier moiety.

e-tag moiety FAM X Pro2  5-FAM C(CH₃)₂ Pro4  5-FAM no carbon Pro6  5-FAM(CH₂)₈ Pro7  5-FAM CH₂OCH₂CH₂OCH₂ Pro8  5-FAMCH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂ Pro9  5-FAM 1,4-phenyl Pro10 6-FAM C(CH₃)₂Pro11 6-FAM no carbon Pro12 6-FAM CH₂OCH₂CH₂OCH₂ Pro13 6-FAMCH₂CH₂0CH₂CH₂0CH₂CH₂0CH₂CH₂Synthesis of Compound 1

To a stirred solution of 5- or 6-carboxyfluorescein (0.5 mmol) in dryDMF (5 mL) were added N-hydroxysuccinimide (1.1 equiv.) and1,3-dicylcohexylcarbodiimide (1.1 equiv.). After about 10 minutes, awhite solid (dicyclohexylurea) started forming. The reaction mixture wasstirred under nitrogen at room temperature overnight. TLC (9:1CH₂Cl₂-MeOH) indicated complete disappearance of the starting material.

The supernatant from the above mixture was added dropwise to a stirredsolution of diamine (2-5 equiv.) in DMF (10 mL). As evident from TLC(40:9:1 CH₂Cl₂-MeOH—H₂O), the reaction was complete instantaneously. Thesolvent was removed under reduced pressure. Flash chromatography of theresulting residue on Iatrobeads silica provided the desired amine(compound 1) in 58-89% yield. The ¹H NMR (300 MHz, DMSO-d₆) of compound1 was in agreement with the assigned structure.

Synthesis of Compound 2

To the amine (compound 1) (0.3 mmol) were sequentially added dry DMF (10mL) and N-succinimidyl iodoacetate (1.1 equiv.). The resulting mixturewas stirred at room temperature until a clear solution was obtained. TLC(40:9:1 CH₂Cl₂-MeOH—H₂O) revealed completion of the reaction.

The above reaction solution was then treated with triethylamine (1.2equiv.) and 3-mercaptopropionic acid (3.2 equiv.). The mixture wasstirred at room temperature overnight. Removal of the solvent underreduced pressure followed by flash chromatography afforded theβ-thioacid (compound 2) in 62-91% yield. The structure of compound 2 wasassigned on the basis of its ¹NMR (300 MHz, DMSO-d₆).

Synthesis of Pro2, Pro4, and Pro6 Through Pro13

To a stirred solution of the β-thioacid (compound 2) (0.05 mmol) in dryDMF (2 mL) were added N-hydroxysuccinimide (1.5 equiv.) and1,3-dicylcohexylcarbodiimide (1.5 equiv.). The mixture was stirred atroom temperature under nitrogen for 24-48 h (until all of the startingmaterial had reacted). The reaction mixture was concentrated underreduced pressure and then purified by flash chromatography to give thetarget molecule in 41-92% yield.

Preparation of Pro1

The compounds of this reaction are shown in FIG. 16B. To a stirredsolution of 5-iodoacetamidofluorescein (compound 4) (24 mg, 0.047 mmol)in dry DMF (2 mL) were added triethylamine (8 μL, 0.057 mmol) and3-mercaptopropionic acid (5□μL, 0.057 mmol). The resulting solution wasstirred at room temperature for 1.5 h. TLC (40:9:1 CH₂Cl₂-MeOH—H₂O)indicated completion of the reaction. Subsequently, N-hydroxysuccinimide(9 mg, 0.078 mmol) and 1,3-dicylcohexylcarbodiimide (18 mg, 0.087 mmol)were added. The reaction mixture was stirred at room temperature undernitrogen for 19 h at which time TLC showed complete disappearance of thestarting material. Removal of the solvent under reduced pressure andsubsequent flash chromatography using 25:1 and 15:1 CH₂Cl₂-MeOH aseluant afforded Pro1 (23 mg, 83%).

Preparation of Pro3

The compounds of this reaction are shown in FIG. 16C. To a stirredsolution of 6-iodoacetamidofluorescein (compound 5) (26 mg, 0.050 mmol)in dry DMF (2 mL) were added triethylamine (8 μL, 0.057 mmol) and3-mercaptopropionic acid (5 μL, 0.057 mmol). The resulting solution wasstirred at room temperature for 1.5 h. TLC (40:9:1 CH₂Cl₂-MeOH—H₂O)indicated completion of the reaction. Subsequently, N-hydroxysuccinimide(11 mg, 0.096 mmol) and 1,3-dicylcohexylcarbodiimide (18 mg, 0.087 mmol)were added. The reaction mixture was stirred at room temperature undernitrogen for 19 h at which time TLC showed complete disappearance of thestarting material. Removal of the solvent under reduced pressure andsubsequent flash chromatography using 30:1 and 20:1 CH₂Cl₂-MeOH aseluant provided Pro3 (18 mg, 61%).

Preparation of Pro5

The compounds of this reaction are shown in FIG. 16D.

Synthesis of Compound 7

To a stirred solution of 5-(bromomethyl) fluorescein (compound 6) (40mg, 0.095 mmol) in dry DMF (5 mL) were added triethylamine (15 μL, 0.108mmol) and 3-mercaptopropionic acid (10□μL, 0.115 mmol). The resultingsolution was stirred at room temperature for 2 days. TLC (40:9:1CH₂Cl₂-MeOH—H₂O) indicated completion of the reaction. The reactionsolution was evaporated under reduced pressure. Finally, flashchromatography employing 30:1 and 25:1 CH₂Cl₂-MeOH as eluant providedthe β-thioacid (compound 7) (28 mg, 66%).

Synthesis of Pro5

To a solution of the acid (compound 7) (27 mg, 0.060 mmol) in dry DMF (2mL) were added N-hydroxysuccinimide (11 mg, 0.096 mmol) and1,3-dicylcohexylcarbodiimide (20 mg, 0.097 mmol). The reaction mixturewas stirred at room temperature under nitrogen for 2 days at which timeTLC (9:1 CH₂Cl₂-MeOH) showed complete disappearance of the startingmaterial. Removal of the solvent under reduced pressure and subsequentflash chromatography with 30:1 CH₂Cl₂-MeOH afforded Pro5 (24 mg, 73%).

Preparation of Pro14

The compounds of this reaction are shown in FIG. 16E.

Synthesis of Compound 9

To 5-aminoacetamidofluorescein (compound 8) (49 mg, 0.121 mmol) weresequentially added dry DMF (4 mL) and N-succinimidyl iodoacetate (52 mg,0.184). A clear solution resulted and TLC (40:9:1 CH₂Cl₂-MeOH—H₂O)indicated complete disappearance of the starting material.

The above reaction solution was then treated with triethylamine (30 μL,0.215 mmol) and 3-mercaptopropionic acid (30□μL, 0.344 mmol). Theresulting mixture was stirred for 2 h. Removal of the solvent underreduced pressure followed by flash chromatography using 20:1 and 15:1CH₂Cl₂-MeOH as eluant gave the β-thioacid (compound 9) (41 mg, 62%). Thestructural assignment was made on the basis of ¹NMR (300 MHz, DMSO-d₆).

Synthesis of Pro14

To a stirred solution of compound 9 (22 mg, 0.04 mmol) in dry DMF (2 mL)were added N-hydroxysuccinimide (9 mg, 0.078 mmol) and1,3-dicylcohexylcarbodiimide (16 mg, 0.078 mmol). The resulting solutionwas stirred at room temperature under nitrogen for about 24 h. Thereaction mixture was concentrated wider reduced pressure and the residuepurified by flash chromatography using 30:1 and 20:1 CH₂Cl₂-MeOH aseluant to give Pro14 (18 mg, 70%).

Synthesis of Pro15, Pro20, Pro22, and Pro28

The synthesis schemes for producing NHS esters of electrophoretic tagsPro15, Pro20, Pro22, and Pro28 are shown in FIGS. 16 F-I, respectively.All of the reagent and reaction conditions are conventional in the artand proceed similarly as the reactions described above.

Example 4 a-tag Reporter Assay for Protein Analysis

A. Labeling of Aminodextran 0/IW 500,000) with an e-tag Moiety andBiotin

Aminodextran was used as a model for demonstrating a-tag reporterrelease in relation to a high molecular weight molecule, which alsoserves as a model for proteins. The number of amino groups for 10 mgaminodextran was calculated as 2×10⁻⁸ moles. For a ratio of 1:4 biotinto e-tag moiety, the number of moles of biotin NHS ester employed was1.85×10⁻⁶, and the number of moles of maleimide NHS ester was 7.4×10⁻⁶.10.9 mg of aminodextran was dissolved in 6 mL of 0.1% PBS buffer. 10 mgof Biotin-x-x NHS ester and 23.7 mg of EMCS were dissolved together in 1mL of DMF and added in 50 μL portions at 30 min intervals to theaminodextran solution while it was stirring and keeping away from thelight. After the final addition of the DMF solution, the mixture waskept overnight (while stirring and away from the light). Then, themixture was dialyzed using a membrane with a molecular weight cut-off of10,000 Daltons. The membrane was immersed in a beaker containing 2 L ofwater while stirring. The water was changed four times in a 2 hinterval. The membrane was kept in the water overnight (while stirringand keeping away from the light). Then the solution was lyophilized andthe lyophilized powder was used for e-tag moiety labeling.

B. Reaction of Biotin and Maleimide Labeled Aminodextran with theMoiety, SAMSA.

SAMSA [5-(((2-(and-3)-S-acetylmercapto)succinoyl)amino)fluorescein] wasemployed as an e-tag moiety to react with maleimide in the aminodextranmolecule. For this purpose 0.3 mg (˜5.3×10⁻⁹ moles) of biotin and EMCSlabeled with aminodextran were dissolved in 10 μl of water. 1.1 mg ofSAMSA (˜1.2×10⁻⁶ moles) was dissolved in 120 μL of 0.1 M NaOH andincubated at room temperature for 15 min (for the activation of thethiol group). Then, the excess of NaOH was neutralized by the additionof 2 μL of 6M HCl, and the pH of the solution was adjusted to 7.0 by theaddition of 30 μL of phosphate buffer (200 mM, pH 7.0). The activatedSAMSA solution was added to the 10 μL solution of the labeledaminodextran and incubated for 1 h. The e-tag moiety-labeledaminodextran was purified with gel filtration using Sephadex G-25(Amersham), and purified samples were collected.

C. The Release of a-tag Reporter from Labeled Aminodextran

2 μL of streptavidin-labeled photosensitizer beads (100 μg/mL) wereadded carefully in the dark to 5 μL of purified labeled aminodextran andincubated in the dark for 15 min. Then the solution was irradiated for 1min at 680 nm. The release of the e-tag reporter was examined be CEusing CE² LabCard™ device (ACLARA BioSciences, Mountain View, Calif.).As shown in FIG. 17A, the CE² LabCard 1 consists of two parts:evaporation control and injection/separation. The evaporation controlincorporates an evaporation control channel 2 (450 μm wide and 50 μmdeep) with two replenishment buffer reservoirs 3 (2 mm in diameter) andthe evaporation-controlled sample well 4 (1 min diameter) in the middleof the evaporation control channel. The volume of the replenishmentbuffer reservoirs are 4.7 μL while the volume of the sample well is only1.2 μL, and the volume of the channel 2 beneath the middle sample wellis about 40 nL. The second part of the CE² device, which is used forinjection and separation, consists of an injection microchannel 5 and aseparation microchannel 6, intersecting at a junction 7, and havingdimensions of 120 μm wide and 50 μm deep. Both ends of the separationchannel and one end or the injection channel connect with bufferreservoirs 8, while the second end of the injection channel connectsdirectly to the evaporation-controlled sample well 4. The channels areenclosed by laminating a film (MT40) to the LabCard™. A detector 9 ispositioned 10 mm from the junction. After filling the CE² LabCard devicewith separation buffer (20 mM HEPES, pH 7.4 and 0.5% PEO), 300 mL of theassay mixture was added to the sample well 4. The sample was injectedinto the microchannel junction 7 by applying voltages to the bufferreservoirs as indicated in FIG. 17B. The sample was then separated as isshown in FIG. 17C.

FIG. 18 shows the electropherograms of purified labeled aminodextranwith and without photosensitizer beads. The addition of thephotosensitizer beads lead to the release of the e-tag reporter from theaminodextran using singlet oxygen produced by photosensitizer upon theirradiation at 680 nm. Experimental conditions: separation buffer 20 mMHEPES pH 7.4, and 0.5% PEO; voltage configurations as described in FIG.17; assay mixture had 29 μg/ml streptavidin-labeled photosensitizerbeads and irradiated for 1 min at 680 nm using 680±10 nm filter and a150 W lamp. As shown, the addition of the photosensitizer beads leads tothe release of the e-tag reporter from the aminodextran using singletoxygen produced by the photosensitizer upon irradiation at 680 nm. Inorder to optimize the irradiation time, reaction mixtures containingphotosensitizer beads were irradiated for different lengths of timeranging from 1 to 10 min. There is no significant increase in the e-tagreporter release for irradiation times longer than 1 min.

FIG. 19 shows the effect of photosensitizer bead concentration on e-tagreporter release. The figure shows the separation of purified labeledaminodextran using different concentrations of photosensitizer beads.The higher concentration of photosensitizer beads leads to the higherrelease of e-tag reporters from the labeled aminodextran. Experimentalconditions: separation buffer 20.0 mM HEPES pH 7.4, and 0.5% PEO;voltage configurations as described for FIG. 17; assay mixture wasirradiated for 1 min at 680 nm using 680±10 nm filter and a 150 W lamp.

FIG. 20 depicts a linear calibration curve for the release of e-tagreporters as a function of photosensitizer bead concentration. Resultswere obtained using a CE² LabCard. Experimental conditions: separationbuffer 20.0 mM HEPES pH 7.4, and 0.5% PEO; voltage configurations asdescribed for FIG. 17; assay mixture was irradiated for 1 min at 680 nmusing 680±10 nm filter and a 150 W lamp.

In addition, the effect of the concentration of labeled aminodextran one-tag reporter release was also examined, with the results shown in FIG.21. As demonstrated in this figure, a lower concentration of labeledaminodextran for a given concentration of photosensitizer beads leads tomore efficient e-tag reporter release (or higher ratio of e-tag reporterreleased to the amount of labeled aminodextran). Results were obtainedusing a CE² LabCard. Experimental conditions: separation buffer 20.0 mMHEPES pH 7.4, and 0.5% PEO; voltage configurations as described for FIG.17; assay mixture had 29 μg/ml of photosensitizer beads and wasirradiated for 1 min at 680 nm using 680±10 nm filter and a 150 W lamp.

Example 5 e-tag Reporter Assay for Protein Analysis

A. Conjugation of e-tag Moieties to Antibodies.

Two different approaches for conjugation were employed. The firstapproach involved the direct attachment of e-tag moieties to theantibody, and the second approach involved attachment of e-tag moietiesto dextran that was then attached to the antibody.

(A1) Direct Conjugation of e-Tag Moieties to Antibodies.

E-tag moieties were synthesized with an-NHS ester end that reacted withprimary amines of the antibody to form a stable amide linkage. Thisresulted in a random attachment of a-tag moieties over the surface ofthe antibody. Modification with up to 6 to 12 NHS ester containingmolecules per antibody molecule typically results in no decrease inantigen binding activity. Even higher ratios of NHS ester to antibodyare possible with only slight loss of activity.

Protocol

1. Purified human IgG (purchased from Sigma-Aldrich) was diluted to 2mg/ml in 1×PBS (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2).

2. NHS ester containing a-tag moieties was dissolved in DMF(dimethylformamide) to a final concentration between 10 to 20 nmols/μlDMF.

3. 500 μL of diluted human IgG (6.5 nmol) was mixed with either 1, 5,25, or 50 μl of a-tag moiety (14, 68, 340, and 680 nmols respectively).

4. The solution was allowed to react for 2 hours on ice in the dark.

5. The e-tag moiety-conjugated antibody was purified by dialysis against0.1×PBS (10 mM sodium phosphate, 15 mM NaCl, pH 7.2) for 20 hours at 4°C.

(A2) Conjugation of a-Tag Moiety-Dextran to Antibodies.

In this second example, a-tag moieties were first attached toamine-containing dextran via an amide linkage essentially as describedabove. Polyclonal and some monoclonal antibodies contain carbohydratesin the Fc portion of the antibody. These polysaccharides can beperiodate oxidizedto form reactive aldehyde residues. The amino-dextrancontaining a-tag moiety is then conjugated to the aldehyde residues ofthe oxidized antibodies through the formation of a Schiff base. Thislinkage is further stabilized by reduction to a secondary amine linkagewith sodium cyanoborohydride.

The extremely large size of the amino-dextran (molecular weight of500,000) containing 50 to 500 available amino-groups for conjugation toa-tag moieties allows for a significant increase in the number of e-tagmoieties per antibody, resulting in signal amplification. Since thedextran is coupled through the carbohydrate on the Fc portion of theantibody, it is sufficiently removed from the antigen-binding sitewithout comprising activity.

Protocol for Conjugation of a-Tag Moieties to Amino-Dextran

1. Amino-dextran (500,000 mw with 500 amines/mole dextran) was dissolvedin 90% DMF to a final concentration of 2 mg/ml (2 nmol amine/μl).

2. NHS ester containing e-tag moieties were dissolved in DMF(dimethylformamide) to a final concentration between 10 to 20 nmols/μlDMF.

3. 500 μl of amino-dextran (1000 nmol of amine) was mixed with either500, 1000, or 2000 nmol a-tag moiety.

4. The solution was allowed to react for 2 hours on ice in the dark.

5. The e-tag moiety-conjugated amino-dextran was purified by dialysisagainst 0.1×PBS (10 mM sodium phosphate, 15 mM NaCl, pH 7.2) for 20hours at 4° C.

6. Precipitate was removed by centrifugation at 14,000×g for 5 minutes.

Protocol for Oxidation of Antibodies with Sodium Periodate

1. 500 μl (2.8 nmol) of purified anti-human IL-4 polyclonal antibody(purchased from Pierce) was oxidized in the presence of 10 mM sodiumperiodate (Aldrich).

2. The solution was allowed to react for 30 minutes at room temperaturein the dark.

3. Ethylene glycol is added to a final concentration of 100 mM andallowed to incubate for 10 minutes at room temperature.

4. The oxidized antibody was purified by dialysis against 0.1×PBS (10 mMsodium phosphate, 15 mM NaCl, pH 7.2) for 2 hours at 4° C.

Protocol for Conjugation of Periodate-Oxidized Antibody to a-TagMoieties Containing Amino-Dextran

1. 54 μl (300 pmol) of oxidized anti-human IL-4 polyclonal antibody ismixed with 300 pmol of a-tag moiety-conjugated amino-dextran in thepresence of 200 mM sodium carbonate, pH 9.5.

2. The solution was allowed to react for 2 hours at room temperature inthe dark.

3. Sodium cyanoborohydride (made fresh in 1 N NaOH) is added to a finalconcentration of 50 mM and allowed to react for 30 minutes at roomtemperature.

4. Unreacted aldehydes are blocked by the addition of 50 mMethanolamine, pH 9.6 and allowed to react for 30 minutes at roomtemperature.

5. The conjugate was purified by dialysis against 0.1×PBS (10 mM sodiumphosphate, 15 mM NaCl, pH 7.2) for 20 hours at 4° C.

B. The Release of a-Tag Reporters from Labeled Aminodextran

The procedure and device employed were substantially as discussed abovefor the SAMSA e-tag moiety. A total of 8 e-tag reporters were separatedusing ABI310. The separation conditions were as follows: 50 microncapillary, 47 cm long and 36 cm end-to-detection; separation buffer,POP-6 (PE Biosystems); injection 60 sec at 3.0 kV; separation voltage,9.4 kV. The results are depicted in the electropherogram of FIG. 22.

C. Immunoassays with Antibodies Conjugated to e-Tag Moieties.

Two types of immunoassays (direct and indirect or sandwich), developedfrom the conjugated e-tag moieties mentioned above, were carried out.

(C1) Sandwich Immunoassays for Cytokines

A sandwich-type immunoassay was carried out (FIG. 23). The assay allowsfor the qualification and quantification of known cytokine antigens. Inthis assay, a matched pair of antibodies forms a sandwich around acytokine antigen bringing the two antibodies in close proximity. One ofthese antibodies is conjugated with an e-tag moiety to yield an e-tagprobe. The e-tag probes have a singlet oxygen labile linkage, whichallows the release of the e-tag reporter after reaction with singletoxygen. The second antibody is conjugated to a photosensitizer dye thatproduces singlet oxygen when irradiated at 680 nm. Due to the relativelyshort half-life of the singlet oxygen, only when the two antibodies forma sandwich does the singlet oxygen cleave the cleavable linkage of thea-tag probe.

Protocol for a Sandwich Immunoassay for Cytokines

1. 10 pl of assay buffer (0.1×PBS, 40 mg/ml BSA) is mixed with 1 pl (100nM) of biotin-labeled anti-human IL-4 monoclonal antibody (purchasedfrom Pierce, catalogue number M-450-B) and 1 μl of cytokine IL-4(Pierce, catalogue number R-IL-4-5) ranging in concentration from 0 to500 nM.

2. The reaction was allowed to proceed for 30 minutes at roomtemperature.

3. 5 μl of 100 μg/ml streptavidin-labeled photosensitizer beads wereadded and the mixture was incubated for 15 minutes at room temperaturein the dark.

4. To remove non-specific interactions of the a-tag probes withstreptavidin, 2 μl of 5 μM biotin-DNP was added and incubated for 10minutes at room temperature in the dark.

5. 1 μl of 400 nM anti-human IL-4 polyclonal antibody conjugated to anamino-dextran e-tag moiety was added and incubated for 30 minutes atroom temperature in the dark.

6. The reaction mixture was then irradiated for 30 s using a 150 wattlamp source with a optical filter of 680 DF±20 nm.

7. 1 μl of ROX T8 standard, 1:20 (from PE Biosystems), is then added andreleased e-tags were separated by capillary electrophoresis either onABI310 or ACLARA plastic LabCard (ACLARA BioSciences, Inc. MountainView, Calif.).

8. Separation conditions of the released a-tag reporters on ABI310 wereas follows: 50 μm capillary, 47 cm long and 36 cm end-to-detection;separation buffer, POP-6; injection 60 s at 3.0 kV; separation Voltage,9.4 kV. The results for IL-4 with the a-tag reporter Pro 1 are shown inFIG. 24.

The above procedure was repeated for various cytokines and various a-tagmoieties as follows: IL-6 was studied using e-tag moiety Pro 10 and theresults are depicted in FIG. 25. IFNγ was studied using e-tag moiety Pro8 and the results are depicted in FIG. 26. TNFα was studied using a-tagmoiety Pro 7 and the results are depicted in FIG. 27. 11-10 was studiedusing a-tag moiety Pro 4 and the results are depicted in FIG. 28. IL-8was studied using a-tag moiety Pro 2 and the results are depicted inFIG. 29.

IL-4 was studied as duplex reactions with other cytokines as follows;IL-4 and IL-6, IL-4 and IL-8, IL-4 and TNFα, and IL-4 and IFNγ. Theresults are depicted in FIG. 30. A multiplexed assay for five cytokines(IL-4, 11-6, IL-8, TNFα, and IFNγ) was carried out and the results aredepicted in FIG. 31. A multiplexed assay for six cytokines (IL-4, IL-6,IL-8, IL-10, TNFα, and IFNγ) was conducted and the results are depictedin FIG. 32.

(C2) Direct Immunoassays for IgG.

In a direct immunoassay, the IgG antigen was conjugated with a-tagmoieties to form e-tag probes. The e-tag probes have a singlet oxygenlabile linkage, which allows the release of the e-tag reporter afterreaction with singlet oxygen. The antibody is conjugated to aphotosensitizer dye that produces singlet oxygen when irradiated at 680nm. Due to the relatively short half-life of the singlet oxygen, onlywhen the two antibodies bind does the singlet oxygen cleave thecleavable linkage to release an a-tag reporter (FIG. 33).

Protocol for Direct Immunoassay for Human IgG

1. 10 μl of assay buffer (0.1×PBS, 40 mg/ml BSA) is mixed with 1 μl (100nM) of biotin-labeled anti-human IgG antibody and 1 μl of human IgG(from Sigma) labeled with an e-tag moiety ranging in concentration from0 to 500 nM.

2. The reaction was allowed to react for 30 minutes at room temperature.

3. 5 μl of 100 μg/ml streptavidin-labeled photosensitizer beads wereadded and the mixture was incubated for 15 minutes at room temperaturein the dark.

4. The reaction mixture was then irradiated for 30 sec using a 150 wattlamp source with a optical filter of 680 DF±20 nm.

5. 1 μl of ROX T8 standard is then added and released e-tag reportersare separated by capillary electrophoresis either on ABI310 or ACLARAplastic LabCard.

The results of various concentrations of human IgG are shown in FIG. 34.A calibration curve is depicted in FIG. 35.

It is evident from the results herein that the subject inventionsprovide powerful ways of preparing compositions for use in multiplexeddeterminations and methods for performing multiplexed determinationsusing such compositions. The methods provide for homogeneous andheterogeneous protocols, with proteins, as exemplary of other classes ofcompounds.

It is further evident from the above results that the subject inventionprovides an accurate, efficient and sensitive process, as well ascompositions for use in the process, to perform multiplexed reactions.The protocols provide for great flexibility in the manner in whichdeterminations are carried out and maybe applied to a wide variety ofsituations involving haptens, antigens, nucleic acids, cells, etc.,where one may simultaneously perform a number of determinations on asingle or plurality of samples and interrogate the samples for aplurality of events. The results of the determination are readily readin a simple manner using electrophoresis or mass spectrometry. Systemsare provided where the entire process, after addition of the sample andreagents, may be performed under the control of a data processor withthe results automatically recorded.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A compound of the formula:T-L-M-D, wherein: T is a polypeptide-binding moiety; L is

wherein * indicates the point of attachment to M, and ** indicates thepoint of attachment to T; M is a divalent moiety comprising up to 100non-hydrogen atoms selected from the group consisting of carbon,nitrogen, oxygen, phosphorus, boron, and sulfur, wherein M contains atleast a group having the structure

D is a fluorescent dye selected from the group consisting of awater-soluble rhodamine dye, a fluorescein, and a4,7-dichlorofluorescein; and wherein *** indicates the point ofattachment directly to an sp2-hybridized carbon atom of the non-fusedbenzene ring of the fluorescent dye.
 2. The compound of claim 1, whereinT comprises an antibody or a fragment thereof.
 3. The compound of claim1, wherein T comprises an antibody specific for a target protein orpolypeptide.
 4. The compound of claim 1, wherein T comprises ahaptenized antibody.
 5. The compound of claim 1, wherein T comprises abiotinylated protein.
 6. The compound of claim 1, wherein T comprises anantibody derivatized with a functional polymer.
 7. The compound of claim1, wherein M comprises a positively charged group.
 8. The compound ofclaim 1, wherein M comprises a negatively charged group.
 9. The compoundof claim 1, wherein M comprises more than one amide group.
 10. Thecompound of claim 9, wherein M is

where *** indicates the point of attachment to D, and * indicates thepoint of attachment to L.
 11. The compound of claim 1, wherein D is aflourescein, where the fluorescein is selected from the group consistingof: 5-carboxyfluorescein, 6-carboxyfluorescein,5-carboxy-4,7-dichlorofluorescein, 6-carboxy-4,7-dichlorofluorescein,2′,7′-dimethoxy-5-carboxy-4,7-dichlorofluorescein,2′,7′-dimethoxy-6-carboxy-4,7-dichlorofluorescein,2′,7′dimethoxy-4′,5′-dichloro-5-carboxyfluorescein,2′,7′dimethoxy-4′,5′-dichloro-6-carboxyfluorescein,2′,7′-dimethoxy-4′,5′-dichloro-5-carboxy-4,7-dichlorofluorescein, 2′,7′dimethoxy-4′,5′-dichloro-6-carboxy-4,7-dichlorofluorescein,1′,2′,7′,8′-dibenzo-5-carboxy-4,7-dichlorofluorescein,1′,2′,7′,8′-dibenzo-6-carboxy-4,7-dichlorofluorescein,1′,2′,7′,8′-dibenzo-4′,5′-dichloro-5-carboxy-4,7-dichlorofluorescein,1′,2′,7′,8′-dibenzo-4′,5′-dichloro-6-carboxy-4,7-dichlorofluorescein,2′,7′-dichloro-5-carboxy-4,7-dichlorofluorescein,2′,7′-dichloro-6-carboxy-4,7-dichlorofluorescein,2′,4′,5′,7′-tetrachloro-5-carboxy-4,7dichlorofluorescein, and2′,4′,5′,7′-tetrachloro-6-carboxy-4,7dichlorofluorescein.
 12. Thecompound of claim 1, wherein D is

and *** indicates the point of attachment to M.